Method for treating epstein-barr virus - positive cancer with immunotherapy

ABSTRACT

A method for predicting whether a patient will be a long-term survivor on treatment of a disease by adoptive cell transfer (ACT), is disclosed comprising: (i) analysing a blood-derived sample obtained from the patient for one or more prognostic markers of long-term survival on treatment of a disease by ACT, and; (ii) based on the analysis of step (i), predicting whether the patient will be a long-term survivor on treatment of the disease by ACT. Also disclosed are methods for treating a patient by ACT, methods for selecting a patient for treatment by ACT, and methods for selecting a patient for treatment of a disease by ACT.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/594,167 entitled “METHOD FOR TREATING EPSTEIN-BARR VIRUS—POSITIVECANCER WITH IMMUNOTHERAPY” filed May 12, 2017, which claims priority toand is a continuation of application PCT/SG2016/050441, filed Sep. 9,2016, which claims priority to Singapore Application No. 10201509979S,filed Dec. 4, 2015, all of which are incorporated herein by reference intheir entirety.

FIELD OF THE INVENTION

The present invention relates to treatment of disease by adoptive celltransfer (ACT), and in particular to the prediction of long-termsurvival on treatment of a disease by ACT based on analysis ofcorrelates of long-term survival in blood or blood-derived samplesobtained from patients.

BACKGROUND OF THE INVENTION

The following description provides a summary of information relevant tothe present disclosure and is not an admission that any of theinformation provided or publications referenced herein is prior art tothe present disclosure.

Epstein-Barr virus (EBV)-positive nasopharyngeal carcinoma (NPC)represents a significant health-care problem for South-East Asia. Theincidence rate of NPC in South-East Asian males is 10 to 21.4 per 100000 (Chang et al., Virus Res (2009) 143: 209-221). Current therapies areeffective in controlling and curing non-metastasized NPC, howevertreatments for metastatic disease are limited. The median survival timefor patients with the disseminated form of disease ranges from 11 to 22months (Wee et al., J Clin Oncol (2005) 23: 6730-6738). An emergingalternative to chemotherapy is the use of immune-therapy strategies,which have focused on increasing the immune response to viral antigens.

Experimental immunotherapies have included dendritic cell (DC)vaccination (Gerdemann et al., Mol Ther (2009) 17: 1616-1625; Chia etal., Ann Oncol (2012) 23: 997-1005; Moosmann et al., Blood (2010) 115:2960-2970), checkpoint inhibitor blockade (NCCT02460224; NCT02339558;Hamid O, Robert C, Daud A, et al. Safety and tumor responses withlambrolizumab (anti-PD-1) in melanoma. N Engl J Med 2013; 369:134-44),cytotoxic-T-lymphocyte (CTL) infusion (Louis et al., Blood (2009) 113:2442-2450; Straathof et al., Blood (2005) 105: 1898-1904; Louis et al.,J Immunother (2010) 33: 983-990) or expansion (Smith et al., Cancer Res.(2012) 72: 1116-1125), and CAR-T therapy. The cellular strategies havethe end objective of inducing a cytotoxic T-cell response that iscapable of directly killing virally infected tumors. Whilst there hasbeen great progress in the application of checkpoint inhibitor blockadeagainst tumors, their use against NPC has been limited. Efficacy of thetherapeutic PD-1 antibody, Pembrolizumab, against NPC resulted in amedian progression free survival rate of 5.6 months (Hamid O, Robert C,Daud A, et al. Safety and tumor responses with lambrolizumab (anti-PD-1)in melanoma. N Engl J Med 2013; 369:134-44). Furthermore, to date therehas been no identification of a reliable biomarker to predict checkpointinhibitor efficacy. CAR-T therapy has shown much promise againstmelanomas and lymphomas but has shown limited efficacy in clearing solidtumors. Similar to checkpoint inhibitor blockade, there has been noclear identification of factors that contribute to this failure.Identification of a series of biomarkers that could identify both howwell patients will respond to therapy and when they will fail to respondwould be invaluable for the healthcare community.

This lack of efficacy of CAR-T therapies against solid tumors has beenhypothesized to be partially attributable to an immunosuppressive statewithin the tumor microenvironment. During the establishment of the tumoran immunosuppressive environment is induced, in which regulatory T cells(Treg) contribute and play a role in the maintenance of this phenotype.Treatment with the chemotherapeutic gemcitabine in a variety of murinecancer models have shown to selectively deplete the Treg populationwithout effecting the CTL population (Suzuki et al., Clin Cancer Res(2005) 11: 6713-6721; Nowak et al., Cancer Res (2002) 62: 2353-2358;Shevchenko et al., Int J Cancer (2013) 133: 98-107). Studies of theeffects of gemcitabine on the Treg compartment in humans have so farbeen limited but early reports are showing that treatment results in asimilar contraction of the regulatory subset both in vitro (Kan et al.,Anticancer Res (2012) 32: 5363-5369) and in vivo (Rettig et al., Int JCancer (2011) 129: 832-838).

Another regulatory cell type that is of increasing interest are themyeloid-derived suppressor cells (MDSCs). MDSCs are a population ofmyeloid cells that are expanded in the presence of various cancers. Inhumans MDSCs are a heterogeneous population but can be broadly definedas HLA-DR−, CD11b+, CD33+, two subsets can be subdivided from thispopulation as either monocytic (CD14+) or granulocytic (CD15+)(Wesolowski et al., J Immunother Cancer (2013)1:10; Dumitru et al.,Cancer Immunol Immunother (2012) 61: 1155-1167; Filipazzi et al., CancerImmunol Immunother (2011) 61: 255-263). These cells have beendemonstrated to be functionally immunosuppressive but their subtype andfrequency is dependent on the disease being studied. Treatment of murinecancer models with gemcitabine shows a reduction in the total number ofMDSCs (Suzuki et al., Clin Cancer Res (2005) 11: 6713-6721; Ding et al.,Cancer Res (2014) 74(13): 3441-3453; Huang et al., Cancer ImmunolImmunother (2013) 62: 1439-1451). The role of MDSC in EBV+NPC is poorlyunderstood.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides method for predictingwhether a patient will be a long-term survivor on treatment of a diseaseby adoptive cell transfer (ACT), comprising:

-   -   (i) analysing a blood-derived sample obtained from the patient        for one or more prognostic markers of long-term survival on        treatment of a disease by ACT, and;    -   (ii) based on the analysis of step (i), predicting whether the        patient will be a long-term survivor on treatment of the disease        by ACT.

In some embodiments, the method comprises analysing the blood-derivedsample for one or more correlates of the size and/or activity ofeffector immune cell and/or immunoregulatory immune cell populations.

In another aspect, the present invention provides a method of treating apatient by adoptive cell transfer (ACT), the method comprising:

-   -   (i) analysing a blood-derived sample obtained from the patient        for one or more prognostic markers of long-term survival on        treatment of a disease by ACT;    -   (ii) based on the analysis of step (ii), predicting long-term        survival of the patient by treatment of the disease by ACT; and    -   (iii) administering one or more doses of cells to the patient.

In another aspect, the present invention provides a method of selectinga patient for treatment of a disease by adoptive cell transfer (ACT),comprising:

-   -   (i) analysing a blood-derived sample obtained from the patient        for one or more prognostic markers of long-term survival on        treatment of a disease by ACT;    -   (ii) based on the analysis of step (i), predicting whether the        patient will be a long-term survivor on treatment of the disease        by ACT; and    -   (iii) selecting a patient predicted to be a long-term survivor        on treatment of the disease by ACT for treatment of the disease        by ACT.

In another aspect, the present invention provides a method of selectinga patient for continued treatment of a disease by adoptive cell transfer(ACT), comprising:

-   -   (i) analysing a blood-derived sample obtained from the patient        for one or more prognostic markers of long-term survival on        treatment of a disease by ACT;    -   (ii) based on the analysis of step (i), predicting whether the        patient will be a long-term survivor on treatment of the disease        by ACT; and    -   (iii) selecting a patient predicted to be a long-term survivor        on treatment of the disease by ACT for continued treatment of        the disease by ACT.

In some embodiments in accordance with various aspects of the presentinvention, the additionally comprises an initial step of administering adose of cells to the patient. In some embodiments, the sample isobtained from the patient within a period of 4 weeks after administeringthe initial step of administering a dose of cells to the patient.

In some embodiments in accordance with various aspects of the presentinvention, the one or more prognostic markers comprise: a marker ofmyeloid-derived suppressor cell (MDSC) number or activity, a marker ofregulatory T lymphocyte number or activity, and/or a marker of effectorT lymphocyte number or activity.

In some embodiments in accordance with various aspects of the presentinvention, the one or more prognostic markers comprise: a marker of theamount of an infectious agent associated with the disease.

In some embodiments in accordance with various aspects of the presentinvention, analysing the blood-derived sample comprises determining oneor more of: the level of interferon gamma (IFNγ), the level of CCL22,the level of IL-10, the level of IL-8, the level of CCL20 and the levelof VEGF.

In some embodiments in accordance with various aspects of the presentinvention, analysing the blood-derived sample comprises:

-   -   (i) determining the ratio of the level of one or more markers of        MDSC number or activity, or the level of one or more markers of        regulatory T lymphocyte number or activity to the level of one        or more markers of effector T lymphocyte number or activity,        and/or    -   (ii) determining the ratio of the level of a marker of the        amount of an infectious agent associated with the disease to the        level of one or more markers of effector T lymphocyte number or        activity.

In some embodiments, the method further comprises determining therelationship between the ratio of (i) and the ratio of (ii).

In some embodiments in accordance with various aspects of the presentinvention, analysing the blood-derived sample comprises: determining thelevel of IFNγ, determining the level of a nucleic acid of an infectiousagent associated with the disease, determining the level of CXCL10,and/or determining the level of CCL20.

In some embodiments, a marker of MDSC number or activity and/orregulatory T lymphocyte number or activity is selected from the groupconsisting of: CXCL10, CCL20, number of MDSCs, the percentage of MDSCsas a proportion of live cells, the number of monocytes, the percentageof monocytes as a proportion of live cells, the percentage of monocytesas a proportion of the number of leukocytes, the percentage ofFoxP3+CTLA4+ regulatory T cells (Tregs) as a proportion of CD3+ cells,myeloid cell marker expression by peripheral blood mononuclear cells(PBMCs), and immune inhibitory factor expression by PBMCs.

In some embodiments, a marker of effector T lymphocyte number oractivity is selected from the group consisting of: IFNγ, lymphocytenumber, T lymphocyte number, the percentage of lymphocytes as aproportion of the number of leukocytes, the percentage of CD8+ cells asa proportion of the number of T lymphocytes, the percentage of CD4+cells as a proportion of the number of T lymphocytes.

In some embodiments, a marker of the amount of an infectious agentassociated with the disease is selected from the group consisting of:viral DNA, viral RNA, viral protein, viral envelope protein.

In some embodiments, in accordance with various aspects of the presentinvention, analysing the blood-derived sample comprises:

-   -   (i) determining the ratio of the level of IFNγ to the level of        CXCL10 and/or CCL20,    -   (ii) determining the ratio of the level of a marker of the        amount of an infectious agent associated with the disease to the        level of IFNγ, and optionally    -   (iii) determining the relationship between the ratio of (i) and        the ratio of (ii).

In a further aspect, the present invention provides a method oftreatment of a disease by adoptive cell transfer (ACT), comprisingadministering to a patient one or more of: an agent for decreasingmyeloid-derived suppressor cell (MDSC) number or activity, an agent fordecreasing regulatory T cell number or activity, an agent for increasingeffector T lymphocyte number or activity, and/or an agent for reducingthe amount of an infectious agent associated with the disease.

In a related aspect, the present invention provides one or more of: anagent for decreasing myeloid-derived suppressor cell (MDSC) number oractivity, an agent for decreasing regulatory T cell number or activity,an agent for increasing effector T lymphocyte number or activity, and/oran agent for reducing the amount of an infectious agent associated withthe disease for use in a method of treatment of a disease by adoptivecell transfer (ACT).

In a further related aspect, the present invention provides the use ofone or more of: an agent for decreasing myeloid-derived suppressor cell(MDSC) number or activity, an agent for decreasing regulatory T cellnumber or activity, an agent for increasing effector T lymphocyte numberor activity, and/or an agent for reducing the amount of an infectiousagent associated with the disease for use in the manufacture of amedicament for use in a method of treatment of a disease by adoptivecell transfer (ACT).

In some embodiments, in accordance with the various aspects, the one ormore agents are administered to the patient prior to treatment by ACT.In some embodiments, the one or more agents are administered to thepatient after a dose of cells has been administered to the patient.

In some embodiments, in accordance with the various aspects of thepresent invention the ACT comprises adoptive transfer of cytotoxic Tlymphocytes (CTLs). In some embodiments, the CTLs are specific for avirus which causes or exacerbates the disease.

In some embodiments, in accordance with the various aspects of thepresent invention the disease is a cancer.

In some embodiments the CTLs are Epstein-Barr Virus (EBV)-specific CTLs.In some embodiments the CTLs are Human papillomavirus (HPV)-specificCTLs.

In some embodiments the cancer is nasopharyngeal carcinoma (NPC),optionally EBV-positive NPC. In some embodiments the cancer is cervicalcancer, optionally HPV-positive cervical cancer.

In a further aspect, the present invention provides a kit comprising:means for detecting a marker of myeloid-derived suppressor cell (MDSC)number or activity and/or a marker of regulatory T lymphocyte number oractivity and means for detecting a marker of effector T lymphocytenumber or activity, optionally further comprising means for detecting amarker of the amount of an infectious agent associated with the disease.

The following paragraphs contain further statements describing thepresent invention:

The inventors demonstrate that the measurement of peripheral seraproteins, in EBV positive NPC patients undergoing CTL therapy after thefirst immunotherapy injection, can accurately identify one- and two-yearsurvivors. Stratification of patients into one-year survivors can beachieved by measurement of eotaxin, MIP-3a (CCL20), and IFNγ, conversionof the measured values into ratios of IFNγ expression, and plotting theratios against each other. Stratification of patients into two-yearsurvivors can be achieved by measurement of EBV DNA, IP10 (CXCL10), andIFNγ, conversion of the measured values into ratios of IFNγ expression,and plotting the ratios against each other.

These parameters can be used to identify patients who will undergosuccessful therapy for the treatment of EBV-positive NPC by adoptivetransfer of EBV-specific CTLs, and thus allow correct powering ofdownstream analysis.

The measurement of monocytic-MDSCs also forms the basis of a prognosticmarker for CTL therapy. Measurement of this cell type can act as amarker for patient stratification. These measurements can have use inother CTL immunotherapies against viral agents.

BRIEF DESCRIPTION OF THE FIGURES

Embodiments and experiments illustrating the principles of the inventionwill now be discussed with reference to the accompanying figures.

FIG. 1. Schematic showing the time points of sample collectionthroughout the Phase II clinical trial.

FIGS. 2A-2D. Graphs showing numbers/percentages of peripheral bloodleukocytes at two weeks after first CTL infusion (i.e. at time point T1)vs. overall survival. FIG. 2A Number of white blood cells, FIG. 2BNeutrophils as a percentage of leukocytes, FIG. 2C Lymphocytes as apercentage of leukocytes, and FIG. 2D Monocytes as a percentage ofleukocytes.

FIG. 3. Table 1 showing the results of statistical analysis ofcorrelation of peripheral blood leukocyte numbers with overall survivalat different time points.

FIGS. 4A-4D. Graphs showing serum levels of cytokines/chemokines and EBVDNA at two weeks after first CTL infusion (i.e. at time point T1) vs.overall survival. FIG. 4A IFNγ, FIG. 4B EBV DNA, FIG. 4C CCL20, and FIG.4D CCL10.

FIG. 5. Table 2 showing the results of statistical analysis ofcorrelation of serum levels of cytokines/chemokines and EBV DNA withoverall survival at different time points.

FIGS. 6A-6B. Graphs showing single analyte analysis of serum levels ofFIG. 6A IFNγ and FIG. 6B EBV DNA at two weeks after first CTL infusion(i.e. at time point T1).

FIGS. 7A-7B. Graphs showing serum analyte ratio analysis at two weeksafter first CTL infusion (i.e. at time point T1). FIG. 7A CCL20:IFNγ vs.EBV DNA:IFNγ, and FIG. 7B CCL10:IFNγ vs. EBV DNA:IFNγ.

FIG. 8. Schematic showing Bayesian network of nanostring analysis of RNAobtained from patient PBMCs at time point T1.

FIG. 9. Table 3 showing the results of statistical analysis ofcorrelation of PBMC transcript level with overall survival at time pointT1.

FIGS. 10A-10C. Graphs showing level of RNA transcripts at two weeksafter first CTL infusion (i.e. at time point T1) vs. overall survival.FIG. 10A CD68, FIG. 10B S100A8, and FIG. 10C LILRA5.

FIGS. 11A-11B. Graphs showing monocyte numbers at different time points.FIG. 11A Monocytes as a percentage of leukocytes, and FIG. 11B Monocytesas a percentage of the total number of live cells.

FIGS. 12A-12B. Graphs showing proportion of cell types as a percentageof leukocytes at different time points. FIG. 12A Neutrophils as apercentage of leukocytes, and FIG. 12B Lymphocytes as a percentage ofleukocytes.

FIG. 13. Scatterplots showing gating strategy for MDSCs for flowcytometry.

FIGS. 14A-14B. Graphs showing number of MDSCs as a percentage of thetotal number of live cells at different time points. FIG. 14A Mean+/−standard deviation for two year survivors and non-survivors, and FIG.14B Individual data points for each patient.

FIG. 15. Graph showing number of FoxP3+CTLA4+ Tregs as a percentage ofCD3+ cells at different time points.

FIGS. 16A-16B. Graphs showing FIG. 16A FoxP3+CTLA4+ Tregs as apercentage of CD3+ cells at T1 in patients with high numbers of MDSCs,and FIG. 16B FoxP3+CTLA4+ Tregs as a percentage of CD3+ cells vs.overall survival.

FIG. 17. Graph showing level of serum cytokines for non-survivors andlong-term survivors (more than 2 years) across all time points.

FIGS. 18A-18C. Graphs showing level of RNA transcripts at T−1(pre-chemotherapy) and T0 (post-chemotherapy) between. FIG. 18A NLRP3,FIG. 18B CD274 (PD-L1), and FIG. 18C STAT4.

FIG. 19. Schematic showing Bayesian network of nanostring analysis ofRNA obtained from patient PBMCs at time point T1.

FIGS. 20A-20C. Graphs showing level of RNA transcripts for differentmarkers at T1 in non-survivors (circles) and long-term survivors (morethan 2 years; squares). FIG. 20A CD68, FIG. 20B LILRA5, and FIG. 20CS100A9.

FIG. 21. Table 4 showing the results of statistical analysis ofcorrelation of PBMC transcript level with survival at time point T1.

FIG. 22. Schematic of Bayesian network of nanostring analysis of RNAobtained from patient PBMCs at time point T0.

FIG. 23. Table 5 showing the results of statistical analysis ofcorrelation of PBMC transcript level with survival at time point T0.

DETAILED DESCRIPTION

The present invention is broadly based on the finding that on the basisof analysis of patients' blood at different time points for correlatesof the size and/or activity of the effector and immunoregulatory cellpopulations, it is possible to predict whether a patient will be along-term survivor on treatment of the disease by adoptive cell transfer(ACT).

Adoptive Cell Transfer

The present invention relates to treatment of disease by adoptive celltransfer (ACT).

ACT involves the transfer of cells into a patient, the cells havingtherapeutic properties for the treatment of the disease. The cellstransferred to the patient may be derived from the patient (i.e.autologous), or may be derived from a different subject (allogenic).

The cells may be immune cells. In some embodiments, the cells may belymphocytes. In some embodiments, the cells may be T lymphocytes.

Adoptive cell transfer of T lymphocytes generally involves obtaining Tcells from a subject, typically by drawing a blood sample from which Tcells are isolated. The T cells are then typically treated or altered insome way, and then administered either to the same subject or to adifferent subject. Adoptive T cell transfer is typically aimed atproviding a T cell population with certain desired characteristics to asubject, or increasing the frequency of T cells with suchcharacteristics in that subject. Adoptive transfer of virus specific Tcells is described, for example, in Cobbold et al., (2005) J. Exp. Med.202: 379-386 and Rooney et al., (1998), Blood 92:1549-1555, herebyincorporated by reference in its entirety.

In some embodiments of the present invention the ACT comprises adoptivetransfer of T cells, e.g. CD4+ or CD8+ T cells.

In some embodiments, the ACT comprises adoptive transfer of T cellswhich are specific for an infectious agent. In some embodiments, the Tcells encode or express a T cell receptor (TCR) which is capable ofrecognising (i.e. binding to) a molecule derived form an infectiousagent, e.g. in the context of presentation by an MHC molecule. In someembodiments, the infectious agent is an agent which is associated withthe disease to be treated by the ACT. In some embodiments, theinfectious agent causes or exacerbates the disease. The infectious agentmay be e.g. a virus, bacteria, fungus, protozoa.

The virus for which the T cells are specific may be a dsDNA virus (e.g.adenovirus, herpesvirus, poxvirus), ssRNA virus (e.g. parvovirus), dsRNAvirus (e.g. reovirus), (+)ssRNA virus (e.g. picornavirus, togavirus),(−)ssRNA virus (e.g. orthomyxovirus, rhabdovirus), ssRNA-RT virus (e.g.retrovirus) or dsDNA-RT virus (e.g. hepadnavirus). The presentdisclosure contemplates viruses of the families adenoviridae,herpesviridae, papillomaviridae, polyomaviridae, poxviridae,hepadnaviridae, parvoviridae, astroviridae, caliciviridae,picornaviridae, coronaviridae, flaviviridae, togaviridae, hepeviridae,retroviridae, orthomyxoviridae, arenaviridae, bunyaviridae, filoviridae,paramyxoviridae, rhabdoviridae and reoviridae.

Viruses associated with a disease or disorder are of particularinterest. Accordingly, the following viruses are contemplated:adenovirus, Herpes simplex type 1 virus, Herpes simplex type 2 virus,Varicella-zoster virus, Epstein-Barr virus, Human cytomegalovirus, Humanherpesvirus type 8, Human papillomavirus, BK virus, JC virus, Smallpox,Hepatitis B virus, Parvovirus B19, Human Astrovirus, Norwalk virus,coxsackievirus, hepatitis A virus, poliovirus, rhinovirus, severe acuterespiratory syndrome virus, hepatitis C virus, yellow fever virus,dengue virus, West Nile virus, TBE virus, Rubella virus, Hepatitis Evirus, Human immunodeficiency virus, influenza virus, lassa virus,Crimean-Congo hemorrhagic fever virus, Hantaan virus, ebola virus,Marburg virus, measles virus, mumps virus, parainfluenza virus,respiratory syncytial virus, rabies virus, hepatitis D virus, rotavirus,orbivirus, coltivirus, and banna virus.

In some embodiments, the virus is Epstein-Barr virus (EBV). Accordingly,in some embodiments the ACT is of EBV-specific T cells (e.g.EBV-specific CTLs). In some embodiments, the EBV is strain B95-8,P3HR-1, or a derivative thereof.

In some embodiments, the virus is Human papillomavirus (HPV).Accordingly, in some embodiments the ACT is of HPV-specific T cells(e.g. HPV-specific CTLs). In some embodiments, the HPV is HPV16 or HPV18or a derivative thereof.

In some embodiments, the T cells are cytotoxic T lymphocytes (CTLs).CTLs are capable of effecting cell death in cells infected with a virusby releasing cytotoxic factors including perforin, granzymes,granulysin, and/or by inducing apoptosis of the infected cell byligating FAS on the infected cell through FASL expressed on the T cell(described for example by Chavez-Galan et al., Cellular and MolecularImmunology (2009) 6(1): 15-25, hereby incorporated by reference in itsentirety). Cytotoxicity can be investigated, for example, using any ofthe methods reviewed in Zaritskaya et al., Expert Rev Vaccines (2011),9(6):601-616, hereby incorporated by reference in its entirety. Oneexample of an assay for cytotoxicity of a T cell for to a target cell isthe ⁵¹Cr release assay, in which target cells are treated with ⁵¹Cr,which they internalise. Lysis of the target cells by T cells results inthe release of the radioactive ⁵¹Cr into the cell culture supernatant,which can be detected.

In some embodiments, the ACT is of EBV-specific CTLs. In someembodiments, the ACT is of HPV-specific CTLs.

Disease to be Treated

The disease to be treated by the ACT may be any disease for which theACT is a therapeutic treatment.

In some embodiments the disease is a disease which is associated with(e.g. caused or exacerbated by) infection with an infectious agent, e.g.an infectious agent described herein. In some embodiments the diseasemay be a disease which is caused or exacerbated by infection with avirus, e.g. a virus described herein.

In some embodiments the disease may be a disease which is associatedwith (e.g. caused or exacerbated by) infection with EBV. Diseasesassociated with and/or caused/exacerbated by EBV infection are describedin Taylor et al., Ann Rev Immunol (2015) 33:787-821 and includenasopharyngeal carcinoma, infectious mononucleosis, Burkitt's lymphoma,Hodgkin's lymphoma, gastric cancer, multiple sclerosis, lymphomatoidgranulomatosis, Gianotti-Crosti syndrome, erythema multiforme, acutegenital ulcers, oral hairy leukoplakia, and disorders related toalpha-synuclein aggregation (e.g. Parkinson's disease, dementia withLewy bodies and multiple system atrophy).

In some embodiments, the disease may be a cancer. In some embodiments,the cancer may be an EBV-positive cancer. A cancer can be determined tobe an EBV-positive cancer by any suitable means, which are well known tothe skilled person.

In some embodiments the cancer is nasopharyngeal carcinoma. In someembodiments the disease is EBV-positive nasopharyngeal carcinoma.

In some embodiments the cancer is hepatic cancer/liver cancer (e.g.hepatocellular carcinoma, hepatoblastoma). In some embodiments thedisease is EBV-positive hepatic cancer/liver cancer.

In some embodiments the cancer is lung cancer (e.g. non-small cell lungcancer (NSCLC)). In some embodiments the disease is EBV-positive lungcancer.

In some embodiments the cancer is gastric cancer (e.g. non-small celllung cancer (NSCLC)). In some embodiments the disease is EBV-positivegastric cancer.

In some embodiments the disease may be a disease which is associatedwith (e.g. caused or exacerbated by) infection with HPV.

Human papillomavirus (HPV) is a DNA virus that establishes productiveinfections in keratinocytes of the skin or mucous membranes. There areover 170 types of HPV, a subset of which HPV types are carcinogenic,including high-risk sexually transmitted types that can develop intogenital neoplasias, including cervical intraepithelial neoplasia (CIN),vulvar intraepithelial neoplasia (VIN), penile intraepithelial neoplasia(PIN), and/or anal intraepithelial neoplasia (AIN), for example.HPV-induced cancers arise when viral sequences are integrated into thecellular DNA of host cellst cells. Some of the HPV “early” genes, suchas E6 and E7, act as oncogenes that promote tumor growth and malignanttransformation.

Diseases associated with and/or caused/exacerbated by HPV infection aredescribed in Doorbar et al. Rev Med Virol (2016); 25: 2-23, and includeCondyloma acuminatum, focal epithelia hyperplasia, cervical neoplasia,anogenital cancers (e.g. cervical cancer (e.g. cervical intraepithelialneoplasia (CIN), LSIL), e.g keratinizing SCC, e.g. of the vulva (e.g.vulvar intraepithelial neoplasia (VIN)), vagina, penis (penileintraepithelial neoplasia (PIN)), anus (anal intraepithelial neoplasia(AIN))), oral papilloma and head and neck cancer (e.g. of the oropharynx(e.g. oropharyngeal carcinoma), head and neck squamous cell carcinoma(HNSCC)).

In some embodiments, the disease may be a cancer. In some embodiments,the cancer may be an HPV-positive cancer, e.g. a HPV16- orHPV18-positive. A cancer can be determined to be an HPV-positive cancerby any suitable means, which are well known to the skilled person.

In some embodiments, the cancer is an anogenital cancer. In someembodiments the disease is a HPV-positive anogenital cancer. In someembodiments the cancer is cervical cancer. In some embodiments thedisease is HPV-positive cervical cancer.

In some embodiments the cancer is a head and neck cancer. In someembodiments the disease is a HPV-positive head and neck cancer. In someembodiments the cancer is oropharyngeal cancer. In some embodiments thedisease is a HPV-positive oropharyngeal cancer. In some embodiments thecancer is HNSCC. In some embodiments the disease is a HPV-positiveHNSCC.

The cancer may be any unwanted cell proliferation (or any diseasemanifesting itself by unwanted cell proliferation), neoplasm or tumor orincreased risk of or predisposition to the unwanted cell proliferation,neoplasm or tumor. The cancer may be benign or malignant and may beprimary or secondary (metastatic). A neoplasm or tumor may be anyabnormal growth or proliferation of cells and may be located in anytissue. Examples of tissues include the adrenal gland, adrenal medulla,anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum,central nervous system (including or excluding the brain) cerebellum,cervix, colon, duodenum, endometrium, epithelial cells (e.g. renalepithelia), gallbladder, oesophagus, glial cells, heart, ileum, jejunum,kidney, lacrimal glad, larynx, liver, lung, lymph, lymph node,lymphoblast, maxilla, mediastinum, mesentery, myometrium, nasopharynx,omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervoussystem, peritoneum, pleura, prostate, salivary gland, sigmoid colon,skin, small intestine, soft tissues, spleen, stomach, testis, thymus,thyroid gland, tongue, tonsil, trachea, uterus, vulva, white bloodcells.

Samples

The present invention involves analysing a sample for one or moreprognostic markers of survival on treatment by ACT.

The sample is obtained from a patient, and may be e.g. a blood or tissuesample. In some embodiments, the sample is a blood or blood-derivedsample. That is, the sample may be whole blood obtained from thepatient, or may be derived from a quantity of blood obtained from thepatient. In some embodiments, a blood derived sample may be quantity ofblood plasma or serum derived from the subject's blood. This maycomprise the fluid portion of the blood obtained after removal of thefibrin clot and blood cells. The sample may be a preparation of cellsobtained from the blood sample (e.g. nucleated cells, PBMCs etc.).

In some embodiments, the sample has been obtained from the subject. Insome embodiments, the method is performed in vitro. In some embodiments,the methods are not practised on the human or animal body.

In some embodiments, the methods comprise a step of obtaining a samplefrom the subject. In some embodiments, the sample may be obtained andthen stored, e.g. at −80° C. The stored sample can be thawed andanalysed in accordance with the methods of the invention.

In some embodiments, a sample is obtained from the patient at apre-determined time point in relation to a proposed or contemporaneouscourse of treatment of the disease. In some embodiments, samples areobtained from the patient at more than one time point in relation to aproposed or contemporaneous course of treatment of the disease.

In some embodiments, the sample is or has been obtained from the patientprior to a therapeutic intervention to treat the disease. A therapeuticintervention to treat the disease may be e.g. chemotherapy,immunotherapy, radiotherapy, surgery, vaccination and/or hormonetherapy. A therapeutic intervention to treat the disease may be ACT,e.g. adoptive transfer of CTLs, e.g. CTLs specific for an infectiousagent which causes or exacerbates the disease.

In some embodiments the sample is or has been obtained from the patientup to 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 3 weeks, 4 weeks, 2months, 3 months, 4 months, 5 months, 6 months or 1 year prior to atherapeutic intervention to treat the disease. In some embodiments thesample is or has been obtained from the patient not more than 1 year, 6months, 5 months, 4 months, 3 months, 2 months, 4 weeks, 3 weeks, 2weeks, 13 days, 12 days, 11 days, 10 days, 9 days, 8 days, 7 days, 6days, 5 days, 4 days, 3 days, 2 days or 1 day prior to a therapeuticintervention to treat the disease.

In some embodiments, the sample is or has been obtained from the patientduring the course of a therapeutic intervention to treat the disease. Insome embodiments, the sample is or has been obtained from the patientafter commencement of a therapeutic intervention to treat the disease.

In some embodiments the sample is or has been obtained from the patientup to 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 3 weeks, 4 weeks, 2months, 3 months, 4 months, 5 months, 6 months or 1 year after atherapeutic intervention to treat the disease (preferably aftercommencement, i.e. first administration of, the therapeuticintervention). In some embodiments the sample is or has been obtainedfrom the patient not more than 1 year, 6 months, 5 months, 4 months, 3months, 2 months, 4 weeks, 3 weeks, 2 weeks, 13 days, 12 days, 11 days,10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 daysor 1 day after a therapeutic intervention to treat the disease(preferably after commencement, i.e. first administration of, thetherapeutic intervention).

In some embodiments, the sample is or has been obtained from the patienton or after completion of the course of a therapeutic intervention totreat the disease, e.g. on or after completion of a course ofchemotherapy or radiotherapy. In some embodiments chemotherapy comprisestreatment with gemcitabine and/or carboplatin.

In some embodiments the sample is or has been obtained from the patientup to 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 3 weeks, 4 weeks, 2months, 3 months, 4 months, 5 months, 6 months or 1 year aftercompletion of the course of a therapeutic intervention to treat thedisease. In some embodiments the sample is or has been obtained from thepatient not more than 1 year, 6 months, 5 months, 4 months, 3 months, 2months, 4 weeks, 3 weeks, 2 weeks, 13 days, 12 days, 11 days, 10 days, 9days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days or 1 dayafter completion of the course of a therapeutic intervention to treatthe disease.

In some embodiments, the sample is or has been obtained from the patientprior to administration of cells to the patient. In some particularembodiments, the sample is or has been obtained from the patient priorto the commencement of treatment of the patient by ACT.

In some embodiments the sample is or has been obtained from the patientafter cells have been administered to the patient. In some embodimentsthe sample is or has been obtained after cells have been administered toa patient in accordance with a method of treatment of the disease byACT. In some embodiments the sample is or has been obtained after cellshave been administered to a patient in accordance with a screening stepto determine whether the patient would be a long-term survivor ontreatment of the disease by ACT.

As used herein, ‘long-term survival’ means survival on treatment of thedisease by ACT of a period of greater than 6 months, e.g. survival for aperiod of greater than 6, 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36months. In some embodiments, long-term survival may be classed assurvival for one of at least 12 months, at least 18 months, at least 24months, or at last 26 months. In some embodiments, long-term survivalmay be classed as survival for at least 24 months.

Survival ‘on treatment’ by ACT means survival during the course oftreatment by administration of cells in accordance with a method oftreating a disease by ACT, e.g. as described herein. A period ofsurvival of a patient on treatment with ACT may be measured from thedate of administration, e.g. first administration, of a therapy to thepatient, e.g. administration of a dose of cells by ACT.

‘Increased’ survival as used herein refers to survival for a period oftime which is greater (i.e. a longer period of time) relative to areference period of time of survival. The reference period may e.g. bethe average (e.g. the mean, median or mode) survival period for apatient on treatment by ACT, or the survival period for a patient who isnot a long-term survivor on treatment of the disease by ACT.

In some embodiments, the sample is or has been obtained from the patientafter administration of a single dose of cells. In some embodiments thesample is or has been obtained after administration of more than onedose of cells, e.g. after 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses of cellshave been administered to the patient.

In some embodiments the sample is or has been obtained from the patientwithin a defined period of time after cells have been administered tothe patient. In some embodiments the sample is or has been obtained fromthe patient up to 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days,8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 3 weeks, 4weeks, 2 months, 3 months, 4 months, 5 months, 6 months or 1 year aftercells have been administered to the patient. In some embodiments thesample is or has been obtained from the patient not more than 1 year, 6months, 5 months, 4 months, 3 months, 2 months, 4 weeks, 3 weeks, 2weeks, 13 days, 12 days, 11 days, 10 days, 9 days, 8 days, 7 days, 6days, 5 days, 4 days, 3 days, 2 days or 1 day after cells have beenadministered to the patient.

In some embodiments the sample is or has been obtained from the patientbetween 1 day to 6 weeks, 1 day to 4 weeks, 5 days to 3 weeks, or 1 weekto 2 weeks after cells have been administered to the patient.

In some embodiments, the methods may comprise analysis of samplesobtained at different time points. For example, in some embodiments themethods may comprise analysis of a sample obtained prior to therapeuticintervention to treat a disease, and analysis after therapeuticintervention to treat a disease.

Analysis of Prognostic Markers

The methods of the present invention involve analysing a sample for oneor more prognostic markers.

The method may be performed in vitro or ex vivo. In some embodiments themethod is not performed on the human or animal body.

The methods comprise analysing blood-derived sample(s) for one or morecorrelates of the size and/or activity of effector immune cell and/orimmunoregulatory immune cell populations.

In some embodiments, the analysis comprises determining the presence of,or level (i.e. the amount) of, a given marker. In some embodiments theanalysis comprises measuring the level of or quantifying the marker.

In some embodiments the method comprises determining thepresence/absence or measuring the level of (e.g. the amount) of a givenmolecule in a sample. In some embodiments, the molecule may be a nucleicacid (e.g. DNA, RNA), protein, peptide, carbohydrate (e.g. a sugar) orlipid.

In some embodiments, the level of a protein may be determined byanalysing the blood-derived sample for nucleic acid encoding theprotein. In some embodiments, the level of a protein may be determinedby analysing the blood-derived sample for the protein or a fragmentthereof. In some embodiments, the level of a protein may be determinedby analysing the blood-derived sample for a correlate of thepresence/activity of the protein.

In some embodiments, the number of a given cell type may be determinedby analysing the blood-derived sample for the expression of a marker orplurality of markers (e.g. nucleic acid/protein/peptide marker(s)) forthat cell type. In some embodiments, the number of a given cell type maybe determined by analysing the blood-derived sample for a correlate ofthe presence/activity of that cell type.

Nucleic acids can be detected and/or measured by various means known tothose skilled in the art. For example, gene expression can be analysedby measuring levels of mRNA by quantitative real-time PCR (qRT-PCR),nanostring analysis etc. RNA/DNA of a pathogen can be measured e.g. byqPCR.

Proteins can be detected and/or measured by various methods well knownin the art, e.g. by antibody-based methods, for example by western blot,immunohistochemistry, immunocytochemistry, flow cytometry, ELISA,ELISPOT, reporter-based methods, etc.

A given activity, e.g. an activity associated with a given cell type,can be measured e.g. by an assay (e.g. an in vitro assay) for thatactivity. In some embodiments, an activity can be measured by analysinga sample for a correlate of the activity, e.g. for a nucleic acid,protein or fragment thereof associated with the activity. By way ofexample, the level of IFNγ protein or RNA/DNA encoding IFNγ is acorrelate of effector T lymphocyte (e.g. CTL) activity.

In some embodiments, the method comprises detecting the presence of, ordetermining the number or proportion of (which may e.g. be expressed asa percentage), a given cell type or class of cells in the sample. Thiscan be achieved by a variety of methods including analysis of cellsobtained from the sample by flow cytometry, e.g. after labelling of thecells with antibodies specific for markers allowing differentiation ofcell types.

The inventors have identified a plurality of markers which arepositively or negatively associated with survival of a patient ontreatment of a disease by ACT, summarised in Table A.

For the avoidance of any doubt, a ‘positive’ association in Table Abelow indicates that an higher number/level/percentage is associatedwith increased and/or long-term survival of a patient on treatment ofthe disease by ACT. A ‘negative’ association in Table A below indicatesthat a lower number/level/percentage is associated with increased and/orlong-term survival of a patient on treatment of the disease by ACT.

TABLE A Positive/negative association with survival on treatment Markerof a disease by ACT Number of myeloid-derived suppressor cell Negative(MDSCs) MDSC activity Negative Number of regulatory T lymphocytesNegative Regulatory T lymphocyte activity Negative Number of lymphocytesPositive Percentage of lymphocytes as a proportion of Positive thenumber of leukocytes Number of effector T lymphocytes Positive EffectorT lymphocyte activity Positive Amount of an infectious agent associatedwith Negative the disease CXCL10 Negative CCL20 Negative CCL22 NegativeIL-10 Negative IL-8 Negative VEGF Negative Nucleic acid of an infectiousagent associated Negative with the disease IFNγ Positive Number ofleukocytes Positive Number of neutrophils Negative Percentage ofneutrophils as a proportion of Negative the number of leukocytes Numberof monocytes Negative Percentage of monocytes as a proportion ofNegative the number of leukocytes Percentage of CD8+ cells as aproportion of Positive the number of T lymphocytes Percentage of CD4+cells as a proportion of Positive the number of T lymphocytes Percentageof monocytes as a proportion of Negative live cells Percentage of MDSCsas a proportion of live Negative cells Percentage of FoxP3+ CTLA4+regulatory T Negative cells (Tregs) as a proportion of CD3+ cellsExpression of myeloid cell markers by Negative peripheral bloodmononuclear cells (PBMCs) Expression of immune inhibitory factors byNegative PBMCs

Accordingly, in some embodiments of the present invention the one ormore prognostic markers of long-term survival on treatment of a diseaseby ACT comprise:

-   -   (a) a marker of MDSC number;    -   (b) a marker of MDSC activity;    -   (c) a marker of regulatory T lymphocyte number;    -   (d) a marker of regulatory T lymphocyte activity;    -   (e) a marker of lymphocyte number;    -   (f) the percentage of lymphocytes as a proportion of the number        of leukocytes;    -   (g) a marker of effector T lymphocyte number;    -   (h) a marker of effector T lymphocyte activity;    -   (i) a marker of the amount of an infectious agent associated        with the disease;    -   (j) the level of CXCL10;    -   (k) the level of CCL20;    -   (l) the level of CCL22;    -   (m) the level of IL-10;    -   (n) the level of IL-8;    -   (o) the level of VEGF;    -   (p) the level of nucleic acid of an infectious agent associated        with the disease;    -   (q) the level of IFNγ;    -   (r) the number of leukocytes;    -   (s) the number of neutrophils;    -   (t) the percentage of neutrophils as a proportion of the number        of leukocytes;    -   (u) the number of monocytes;    -   (v) the percentage of monocytes as a proportion of the number of        leukocytes;    -   (w) the percentage of CD8+ cells as a proportion of the number        of T lymphocytes;    -   (x) the percentage of CD4+ cells as a proportion of the number        of T lymphocytes;    -   (y) the percentage of monocytes as a proportion of live cells;    -   (z) the percentage of myeloid derived suppressor cells (MDSCs)        as a proportion of live cells;    -   (aa) the percentage of FoxP3+CTLA4+ regulatory T cells (Tregs)        as a proportion of CD3+ cells;    -   (bb) the level of expression of one or more myeloid cell markers        by peripheral blood mononuclear cells (PBMCs); and/or    -   (cc) the level of expression of one or more immune inhibitory        factors by PBMCs.

In some embodiments, the methods comprise analysing a blood-derivedsample obtained from a patient for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, or 19 prognostic markers of long-termsurvival on treatment of a disease by ACT.

In some embodiments of the present invention, analysing theblood-derived sample comprises one or more of:

-   -   (a) determining myeloid-derived suppressor cell (MDSC) number;    -   (b) determining the level of myeloid-derived suppressor cell        (MDSC) activity;    -   (c) determining the regulatory T lymphocyte number;    -   (d) determining the level of regulatory T lymphocyte activity;    -   (e) determining lymphocyte number;    -   (f) determining the percentage of lymphocytes as a proportion of        the number of leukocytes;    -   (g) determining effector T lymphocyte number;    -   (h) determining the level of effector T lymphocyte activity;    -   (i) determining the amount of an infectious agent associated        with the disease;    -   (j) determining the level of CXCL10;    -   (k) determining the level of CCL20;    -   (l) determining the level of CCL22;    -   (m) determining the level of IL-10;    -   (n) determining the level of IL-8;    -   (o) determining the level of VEGF;    -   (p) determining the level of nucleic acid of an infectious agent        associated with the disease;    -   (q) determining the level of IFNγ;    -   (r) determining the number of leukocytes;    -   (s) determining the number of neutrophils;    -   (t) determining the percentage of neutrophils as a proportion of        the number of leukocytes;    -   (u) determining the number of monocytes;    -   (v) determining the percentage of monocytes as a proportion of        the number of leukocytes;    -   (w) determining the percentage of CD8+ cells as a proportion of        the number of T lymphocytes;    -   (x) determining the percentage of CD4+ cells as a proportion of        the number of T lymphocytes;    -   (y) determining the percentage of monocytes as a proportion of        live cells;    -   (z) determining the percentage of myeloid derived suppressor        cells (MDSCs) as a proportion of live cells;    -   (aa) determining the percentage of FoxP3+CTLA4+ regulatory T        cells (Tregs) as a proportion of CD3+ cells;    -   (bb) determining the level of expression of one or more myeloid        cell markers by peripheral blood mononuclear cells (PBMCs);        and/or    -   (cc) determining the level of expression of one or more immune        inhibitory factors by PBMCs.

In some embodiments, the one or more prognostic markers comprise: amarker of myeloid-derived suppressor cell (MDSC) number or activity, amarker of regulatory T lymphocyte number or activity, and/or a marker ofeffector T lymphocyte number or activity. In some embodiments, the oneor more prognostic markers comprise: a marker of the amount of aninfectious agent associated with the disease.

MDSC number or activity may be determined by analysis of one or moreproperties of MDSCs described, for example, in Greten et al., Int.Immunopharmacol (2011) 11:802-807, and Gabrilovich and Nagaraj, Nat RevImmunol (2009) 9:162-174, both hereby incorporated by reference in theirentirety. In some embodiments, MDSCs may be identified by reference toone or more of the following surface markers: CD33+, CD11b+, CD14+,CD16−, CD15−, HLADR^(low).

In some embodiments, a marker of myeloid-derived suppressor cell (MDSC)number or activity, or a marker of regulatory T lymphocyte number oractivity, may be selected from the group consisting of: CXCL10, CCL20,CCL22, IL-10, IL-8, number of MDSCs, the percentage of MDSCs as aproportion of live cells, the number of monocytes, the percentage ofmonocytes as a proportion of live cells, the percentage of monocytes asa proportion of the number of leukocytes, myeloid cell marker expressionby peripheral blood mononuclear cells (PBMCs), immune inhibitory factorexpression by PBMCs and the percentage of FoxP3+CTLA4+ regulatory Tcells (Tregs) as a proportion of CD3+ cells.

In some embodiments, a myeloid cell marker and/or an immune inhibitoryfactor/marker according to the present invention may be selected fromTable 3 (FIG. 9). In some embodiments, the myeloid cell marker and/or animmune inhibitory factor/marker may be selected from one or more ofCD68, LILRA5, S100A8, S100A9, S100A12, LILRB4, MSR1, CXCL16, CLEC7A andTREM1. In some embodiments, the myeloid cell marker and/or an immuneinhibitory factor/marker may be selected from one or more of CD68,LILRA5, S100A8 and S100A9.

In some embodiments, a myeloid cell marker and/or an immune inhibitoryfactor/marker may be selected from one or more of NLRP3, PD-L1 andSTAT4.

In some embodiments, a myeloid cell marker and/or an immune inhibitoryfactor/marker according to the present invention may be selected fromone or more of IL-10, CCL22, IL-IL-8, VEGF and CCL20.

T lymphocyte number or activity may be determined by analysis of one ormore properties of T lymphocytes which are well known to the skilledperson. T lymphocyte markers include CD3 polypeptides (e.g. CD3γ CD3εCD3ζ or CD3δ), TCR polypeptides (TCRα or TCRβ), CD27, CD28, CD4 and CD8.

As used herein, a ‘regulatory T lymphocyte’ refers to a T lymphocytehaving immunomodulatory activity. Regulatory T lymphocytes (Tregs)generally suppress/downregulate induction, proliferation and/or functionof effector T cells. Treg phenotype and function is reviewed by Vignaliet al., Nat Rev Immunol (2008) 8(7):523-532, which is herebyincorporated by reference in its entirety. Markers of Tregs includeFoxP3 and CTLA4 expression.

As used herein, an ‘effector T lymphocyte’ refers to T lymphocyteshaving an effector function. Examples of effector T lymphocytes (Teffs)include T helper cells, cytotoxic T lymphocytes (CTLs) and memory Tcells. Teff phenotype and function is reviewed in Janeway,Immunobiology: The Immune System in Health and Disease; 5^(th) Edn.2001, at chapter 8 (hereby incorporated by reference in its entirety).

In some embodiments, a marker of effector T cell number or activity maybe selected from the group consisting of: IFNγ, lymphocyte number, Tlymphocyte number, the percentage of lymphocytes as a proportion of thenumber of leukocytes, effector T lymphocyte number, the percentage ofCD8+ cells as a proportion of the number of T lymphocytes, and thepercentage of CD4+ cells as a proportion of the number of T lymphocytes.

The amount of an infectious agent associated with the disease can bedetermined by analysis e.g. of the amount of a marker of the infectiousagent. For example, a marker of the infectious agent may be a nucleicacid (e.g. RNA or DNA) of the infectious agent or a protein/peptide ofthe infectious agent. In some embodiments, a marker of the amount of aninfectious agent associated with the disease is selected from the groupconsisting of: viral DNA (e.g. viral genomic DNA), viral RNA (e.g. viralgenomic RNA), viral protein, and viral envelope protein.

In some embodiments, analysing the blood-derived sample comprises:determining the level of IFNγ, determining the level of a nucleic acidof an infectious agent associated with the disease, determining thelevel of CXCL10, and/or determining the level of CCL20.

In some embodiments, analysing the blood-derived sample comprisesdetermining one or more ratios between the determined levels ofprognostic markers of long-term survival on treatment of a disease byACT. Levels and/or ratios may be transformed (e.g. log₁₀, or log_(e)transformed) to assist the analysis.

In some embodiments, the analysis comprises determining the ratio of thelevel of a marker of the amount of an infectious agent associated withthe disease to the level of one or more markers of MSDC number oractivity and/or one or more markers of regulatory T lymphocyte number oractivity, and/or determining the ratio of the level of a marker of theamount of an infectious agent associated with the disease to the levelof one or more markers of effector T lymphocyte number or activity.

In some embodiments, the analysis comprises determining the ratio of thelevel of one or more molecules derived from an infectious agentassociated with the disease to the level of one or more factors producedby MDSCs and/or one or more factors produced by regulatory Tlymphocytes, and/or determining the ratio of the level of one or moremolecules derived from an infectious agent associated with the diseaseto the level of one or more factors produced by effector T lymphocytes.

In some embodiments, analysing the blood-derived sample comprises:determining the ratio of the level of CCL20 to the level of IFNγ,determining the ratio of the level of CXCL10 to the level of IFNγ,and/or determining the ratio of the level of a nucleic acid of aninfectious agent associated with the disease to the level of IFNγ.

In some embodiments of the methods of the present invention, analysingthe blood-derived sample comprises comparing the determinedlevel/number/percentage of one or more prognostic markers of long-termsurvival to reference value(s) for the marker. In some embodiments, thereference value is a value for that marker which is indicative oflong-term survival or non-survival of a patient on treatment of adisease by ACT.

In some embodiments the reference value is the value for that marker ina comparable sample obtained from a subject of interest. In someembodiments, the subject is a healthy control individual. In someembodiments the subject is patient having the disease. In someembodiments the subject is a patient surviving for a longer than average(e.g. median or mean, mode) amount of time on treatment of the diseaseby ACT (i.e. a patient surviving for a longer amount of time than theaverage survival of a patient on treatment of the disease by ACT). Insome embodiments the subject is a long-term survivor on treatment of thedisease by ACT. In some embodiments the subject is a patient survivingfor a shorter than average amount of time on treatment of the disease byACT (i.e. a patient surviving for a shorter amount of time than theaverage survival of a patient on treatment of the disease by ACT). Insome embodiments the subject is not a long-term survivor on treatment ofthe disease by ACT.

In some embodiments the value may be an average (e.g. mean, median,mode) value for the marker for a plurality of subjects, e.g. a pluralityof subjects according to an embodiment of the preceding paragraph.

In some embodiments, determination of one or more of the following inrelation to a reference value for a patient surviving for shorter thanthe average amount of time on treatment of the disease by ACT, a patientwho is not a long-term survivor on treatment of the disease by ACT, or apatient surviving for the average amount of time on treatment of thedisease by ACT may be predictive of increased and/or long-term survivalon treatment of the disease by ACT:

-   -   (1) lower number of MDSCs;    -   (2) lower MDSC activity;    -   (3) lower number of regulatory T lymphocytes;    -   (4) lower regulatory T lymphocyte activity;    -   (5) greater number of lymphocytes;    -   (6) greater percentage of lymphocytes as a proportion of the        number of leukocytes;    -   (7) greater number of effector T lymphocytes;    -   (8) greater effector T lymphocyte activity;    -   (9) lower amount of an infectious agent associated with the        disease;    -   (10) lower CXCL10;    -   (11) lower CCL20;    -   (12) lower CCL22;    -   (13) lower IL-10;    -   (14) lower IL-8;    -   (15) lower VEGF;    -   (16) lower amount of nucleic acid of an infectious agent        associated with the disease;    -   (17) greater IFNγ;    -   (18) greater number of leukocytes;    -   (19) lower number of neutrophils;    -   (20) lower percentage of neutrophils as a proportion of the        number of leukocytes;    -   (21) lower number of monocytes;    -   (22) lower percentage of monocytes as a proportion of the number        of leukocytes;    -   (23) greater percentage of CD8+ cells as a proportion of the        number of T lymphocytes;    -   (24) greater percentage of CD4+ cells as a proportion of the        number of T lymphocytes;    -   (25) lower percentage of monocytes as a proportion of the number        of live cells;    -   (26) lower percentage of MDSCs as a proportion of the number of        live cells;    -   (27) lower percentage of FoxP3+CTLA4+ Tregs as a proportion of        the number of CD3+ cells;    -   (28) lower level of expression of one or more myeloid cell        markers by PBMCs; and/or    -   (29) lower level of expression of one or more immune inhibitory        factors by PBMCs.

In some embodiments, determination of one or more of the following inrelation to a reference value for a patient surviving for longer thanthe average amount of time on treatment of the disease by ACT, or along-term survivor on treatment of the disease by ACT, may be predictiveof increased and/or long-term survival on treatment of the disease byACT:

-   -   (1) comparable or lower number of MDSCs;    -   (2) comparable or lower MDSC activity;    -   (3) comparable or lower number of regulatory T lymphocytes;    -   (4) comparable or lower regulatory T lymphocyte activity;    -   (5) comparable or greater number of lymphocytes;    -   (6) comparable or greater number of lymphocytes as a proportion        of the number of leukocytes;    -   (7) comparable or greater number of effector T lymphocytes;    -   (8) comparable or greater effector T lymphocyte activity;    -   (9) comparable or lower amount of an infectious agent associated        with the disease;    -   (10) comparable or lower CXCL10;    -   (11) comparable or lower CCL20;    -   (12) comparable or lower CCL22;    -   (13) comparable or lower IL-10;    -   (14) comparable or lower IL-8;    -   (15) comparable or lower VEGF;    -   (16) comparable or lower amount of nucleic acid of an infectious        agent associated with the disease;    -   (17) comparable or greater IFNγ;    -   (18) comparable or greater number of leukocytes;    -   (19) comparable or lower number of neutrophils;    -   (20) comparable or lower percentage of neutrophils as a        proportion of the number of leukocytes;    -   (21) comparable or lower number of monocytes; (22) comparable or        lower percentage of monocytes as a proportion of the number of        leukocytes;    -   (23) comparable or greater percentage of CD8+ cells as a        proportion of the number of T lymphocytes;    -   (24) comparable or greater percentage of CD4+ cells as a        proportion of the number of T lymphocytes;    -   (25) comparable or lower percentage of monocytes as a proportion        of the number of live cells;    -   (26) comparable or lower percentage of MDSCs as a proportion of        the number of live cells;    -   (27) comparable or lower percentage of FoxP3+CTLA4+ Tregs as a        proportion of the number of CD3+ cells;    -   (28) comparable or lower level of expression of one or more        myeloid cell markers by PBMCs; and/or    -   (29) comparable or lower level of expression of one or more        immune inhibitory factors by PBMCs.

In some embodiments, the sample for such analysis is/has been obtainedfrom a patient after a dose (e.g. a first dose) of cells have beenadministered to the patient by ACT, e.g. within 4 weeks ofadministration of the cells. In some embodiments, the sample for suchanalysis is/has been obtained from a patient prior to a therapeuticintervention (e.g. chemotherapy and/or ACT) to treat the disease. Insome embodiments, the sample for such analysis is/has been obtained froma patient after completion of a therapeutic intervention (e.g.chemotherapy) to treat the disease, and prior to administration of cellsto the patient.

In some embodiments, a lower level/number/percentage as compared to areference value is one of less than 1 times, less than 0.95 times, lessthan 0.9 times, less than 0.85 times, less than 0.8 times, less than0.75 times, less than 0.7 times, less than 0.65 times, less than 0.6times, less than 0.55 times, less than 0.5 times, less than 0.45 times,less than 0.4 times, less than 0.35 times, less than 0.3 times, lessthan 0.25 times, less than 0.2 less than 0.15 times, or less than 0.1times the level/number/percentage of the reference value.

In some embodiments, a greater level/number/percentage as compared to areference value is one of more than 1 times, more than 1.1 times, morethan 1.2 times, more than 1.3 times, more than 1.4 times, more than 1.5times, more than 1.6 times, more than 1.7 times, more than 1.8 times,more than 1.9 times, more than 2 times, more than 2.1 times, more than2.2 times, more than 2.3 times, more than 2.4 times, more than 2.5times, more than 2.6 times, more than 2.7 times, more than 2.8 times,more than 2.9 times, more than 3 times, more than 3.1 times, more than3.2 times, more than 3.3 times, more than 3.4 times, more than 3.5times, more than 3.6 times, more than 3.7 times, more than 3.8 times,more than 3.9 times, more than 4 times, more than 4.1 times, more than4.2 times, more than 4.3 times, more than 4.4 times, more than 4.5times, more than 4.6 times, more than 4.7 times, more than 4.8 times,more than 4.9 times, or more than 5 times the level/number/percentage ofthe reference value.

In some embodiments, a number of leukocytes of one of about 7×10⁹/L,6.5×10⁹/L, 6×10⁹/L, 5.5×10⁹/L, 5×10⁹/L, 4.5×10⁹/L, 4×10⁹/L or lower ispredictive of increased and/or long-term survival. In some embodiments,the sample for such analysis is/has been obtained from a patient after adose (e.g. a first dose) of cells have been administered to the patientby ACT, e.g. within 4 weeks of administration of the cells.

In some embodiments, a percentage of neutrophils as a proportion ofleukocytes one of about 70%, 65%, 60%, 55%, 50%, 45%, 40% or lower ispredictive of increased and/or long-term survival, e.g. in a sampleobtained from a patient after a dose (e.g. a first dose) of cells havebeen administered to the patient by ACT, e.g. within 4 weeks ofadministration of the cells.

In some embodiments, a percentage of lymphocytes as a proportion ofleukocytes of one of about 10%, 15%, 20%, 25%, 30%, 35%, 40% or higheris predictive of increased and/or long-term survival, e.g. in a sampleobtained from a patient after a dose (e.g. a first dose) of cells havebeen administered to the patient by ACT, e.g. within 4 weeks ofadministration of the cells.

In some embodiments, a log 10 transformed level of IFNγ of one of about0.25 pg/ml, 0.3 pg/ml, 0.35 pg/ml, 0.4 pg/ml, 0.45 pg/ml, 0.5 pg/ml,0.55 pg/ml, 0.6 pg/ml, 0.65 pg/ml, 0.7 pg/ml, 0.75 pg/ml, 0.8 pg/ml,0.85 pg/ml, 0.9 pg/ml, 0.95 pg/ml, or 1.0 pg/ml or higher is predictiveof increased and/or long-term survival, e.g. in a sample obtained from apatient after a dose (e.g. a first dose) of cells have been administeredto the patient by ACT, e.g. within 4 weeks of administration of thecells.

In some embodiments, a log 10 transformed level of EBV DNA of one ofabout 4.0 pg/ml, 3.5 pg/ml, 3.0 pg/ml, 2.5 pg/ml, 2.0 pg/ml, 1.5 pg/mlor lower is predictive of increased and/or long-term survival, e.g. in asample obtained from a patient after a dose (e.g. a first dose) of cellshave been administered to the patient by ACT, e.g. within 4 weeks ofadministration of the cells, e.g. in a sample obtained from a patientprior to a therapeutic intervention (e.g. chemotherapy and/or ACT) totreat the disease, or a sample obtained from a patient after completionof a therapeutic intervention (e.g. chemotherapy) to treat the disease,and prior to administration of cells to the patient.

In some embodiments, a log 10 transformed level of CCL20 of one of about1.0 pg/ml, 0.9 pg/ml, 0.8 pg/ml, 0.7 pg/ml, 0.6 pg/ml, 0.5 pg/ml, 0.4pg/ml, 0.3 pg/ml, 0.2 pg/ml, 0.1 pg/ml or lower is predictive ofincreased and/or long-term survival, e.g. in a sample obtained from apatient after a dose (e.g. a first dose) of cells have been administeredto the patient by ACT, e.g. within 4 weeks of administration of thecells.

In some embodiments, a log 10 transformed level of CCL22 of one of about2.0 pg/ml, 1.9 pg/ml, 1.8 pg/ml, 1.7 pg/ml, 1.6 pg/ml, 1.5 pg/ml, orlower is predictive of increased and/or long-term survival.

In some embodiments, a log 10 transformed level of IL-10 of one of about0.5 pg/ml, 0.4 pg/ml, 0.3 pg/ml, 0.2 pg/ml, 0.1 pg/ml, or lower ispredictive of increased and/or long-term survival.

In some embodiments, a log 10 transformed level of IL-8 of one of about1.0 pg/ml, 0.9 pg/ml, 0.8 pg/ml, 0.7 pg/ml, 0.6 pg/ml, 0.5 pg/ml, 0.4pg/ml, 0.3 pg/ml, 0.2 pg/ml, 0.1 pg/ml, or lower is predictive ofincreased and/or long-term survival.

In some embodiments, a log 10 transformed level of VEGF of one of about2.0 pg/ml, 1.9 pg/ml, 1.8 pg/ml, 1.7 pg/ml, 1.6 pg/ml, 1.5 pg/ml, 1.4pg/ml, 1.3 pg/ml, 1.2 pg/ml, 1.1 pg/ml, 1.0 pg/ml or lower is predictiveof increased and/or long-term survival.

In some embodiments, a log 10 transformed level of CXCL10 of one ofabout 2.6 pg/ml, 2.55 pg/ml, 2.5 pg/ml, 2.45 pg/ml, 2.4 pg/ml, 2.35pg/ml, 2.3 pg/ml, 2.25 pg/ml, 2.2 pg/ml, 2.15 pg/ml, 2.1 pg/ml, 2.05pg/ml, 2.0 pg/ml or lower is predictive of increased and/or long-termsurvival, e.g. in a sample obtained from a patient after a dose (e.g. afirst dose) of cells have been administered to the patient by ACT, e.g.within 4 weeks of administration of the cells.

In some embodiments, a ratio of the log 10 transformed level of EBV DNAto the log 10 transformed level of IFNγ of one of less than about 4.0,3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6,2.5, 2.4, 2.3, 2.2, 2.1, or 2.0 is predictive of increased and/orlong-term survival, e.g. in a sample obtained from a patient after adose (e.g. a first dose) of cells have been administered to the patientby ACT, e.g. within 4 weeks of administration of the cells. In someembodiments a ratio of the log 10 transformed level of EBV DNA to thelog 10 transformed level of IFNγ of less than about 3.0 is predictive ofincreased and/or long-term survival.

In some embodiments, a ratio of the log 10 transformed level of CCL20 tothe log 10 transformed level of IFNγ of one of less than about 2.0, 1.9,1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5,0.4, 0.3, 0.2, or 0.1 is predictive of increased and/or long-termsurvival, e.g. in a sample obtained from a patient after a dose (e.g. afirst dose) of cells have been administered to the patient by ACT, e.g.within 4 weeks of administration of the cells.

In some embodiments, a ratio of the log 10 transformed level of CXCL10to the log 10 transformed level of IFNγ of one of less than about 3.0,2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6,1.5, 1.4, 1.3, 1.2, 1.1 or 1.0 is predictive of increased and/orlong-term survival, e.g. in a sample obtained from a patient after adose (e.g. a first dose) of cells have been administered to the patientby ACT, e.g. within 4 weeks of administration of the cells. In someembodiments a ratio of the log 10 transformed level of CXCL10 to the log10 transformed level of IFNγ of less than about 2.5 is predictive ofincreased and/or long-term survival.

In some embodiments, a ratio of the log 10 transformed level of EBV DNAto the log 10 transformed level of IFNγ of one of less than about 4.0,3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6,2.5, 2.4, 2.3, 2.2, 2.1, or 2.0 and a ratio of the log 10 transformedlevel of CCL20 to the log 10 transformed level of IFNγ of one of lessthan about 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9,0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 is predictive of increasedand/or long-term survival, e.g. in a sample obtained from a patientafter a dose (e.g. a first dose) of cells have been administered to thepatient by ACT, e.g. within 4 weeks of administration of the cells.

In some embodiments, a ratio of the log 10 transformed level of EBV DNAto the log 10 transformed level of IFNγ of one of less than about 4.0,3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6,2.5, 2.4, 2.3, 2.2, 2.1, or 2.0 and a ratio of the log 10 transformedlevel of CXCL10 to the log 10 transformed level of IFNγ of one of lessthan about 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9,1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1 or 1.0 is predictive of increasedand/or long-term survival, e.g. in a sample obtained from a patientafter a dose (e.g. a first dose) of cells have been administered to thepatient by ACT, e.g. within 4 weeks of administration of the cells. Insome embodiments a ratio of the log 10 transformed level of EBV DNA tothe log 10 transformed level of IFNγ of less than about 3.0 and a ratioof the log 10 transformed level of CXCL10 to the log 10 transformedlevel of IFNγ of less than about 2.5 is predictive of increased and/orlong-term survival.

In some embodiments, a normalised count of the number of copies of CD68RNA (e.g. as determined by nanostring analysis) of about 200, 195, 190,185, 180, 175, 170, 165, 160, 155, 150, 145, 140, 135, 130, 125, 120,115, 110, 105, 100, 95, 90 or lower is predictive of increased and/orlong-term survival, e.g. in a sample obtained from a patient after adose (e.g. a first dose) of cells have been administered to the patientby ACT, e.g. within 4 weeks of administration of the cells.

In some embodiments, a normalised count of the number of copies ofS100A8 RNA (e.g. as determined by nanostring analysis) of about 3500,3250, 3000, 2750, 2500, 2250, 2000, 1750, 1500, 1250, 1000 or lower ispredictive of increased and/or long-term survival, e.g. in a sampleobtained from a patient after a dose (e.g. a first dose) of cells havebeen administered to the patient by ACT, e.g. within 4 weeks ofadministration of the cells.

In some embodiments, a normalised count of the number of copies ofS100A9 RNA (e.g. as determined by nanostring analysis) of about 4500,4250, 4000, 3750, 3500, 3250, 3000, 2750, 2500, 2250, 2000, 1750, 1500,1250, 1000 or lower is predictive of increased and/or long-termsurvival, e.g. in a sample obtained from a patient after a dose (e.g. afirst dose) of cells have been administered to the patient by ACT, e.g.within 4 weeks of administration of the cells.

In some embodiments, a normalised count of the number of copies ofLILR5A RNA (e.g. as determined by nanostring analysis) of about 90, 85,80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30 or lower is predictive ofincreased and/or long-term survival, e.g. in a sample obtained from apatient after a dose (e.g. a first dose) of cells have been administeredto the patient by ACT, e.g. within 4 weeks of administration of thecells.

In some embodiments, a percentage of monocytes as a proportion ofleukocytes of one of fewer than 15%, 14%, 13%, or 12% is predictive ofincreased and/or long-term survival, e.g. in a sample from a patientprior to a therapeutic intervention (e.g. chemotherapy and/or ACT) totreat the disease, or a sample obtained from a patient after completionof a therapeutic intervention (e.g. chemotherapy) to treat the disease,and prior to administration of cells to the patient.

In some embodiments, a percentage of monocytes as a proportion of livecells is of one of fewer than 70%, 65%, 60%, 55%, 50%, or 45% ispredictive of increased and/or long-term survival, e.g. in a sampleobtained from a patient prior to a therapeutic intervention (e.g.chemotherapy and/or ACT) to treat the disease, or a sample obtained froma patient after completion of a therapeutic intervention (e.g.chemotherapy) to treat the disease, and prior to administration of cellsto the patient.

In some embodiments, a percentage of monocytes as a proportion of livecells is of one of fewer than 60%, 55%, 50%, 45%, 40%, or 35% ispredictive of increased and/or long-term survival, e.g. in a sampleobtained from a patient after a dose (e.g. a first dose) of cells havebeen administered to the patient by ACT, e.g. within 4 weeks ofadministration of the cells.

In some embodiments, a percentage of neutrophils as a proportion ofleukocytes of one of fewer than 80%, 75%, 70%, or 65% is predictive ofincreased and/or long-term survival, e.g. in a sample obtained from apatient after a dose (e.g. a first dose) of cells have been administeredto the patient by ACT, e.g. within 4 weeks of administration of thecells.

In some embodiments, a number of lymphocytes as a proportion of thenumber of leukocytes of one of greater than 15%, 16%, 17%, 18%, 19%, or20% is predictive of increased and/or long-term survival, e.g. in asample obtained from a patient after completion of a therapeuticintervention (e.g. chemotherapy) to treat the disease, and prior toadministration of cells to the patient, or a sample obtained from apatient after a dose (e.g. a first dose) of cells have been administeredto the patient by ACT, e.g. within 4 weeks of administration of thecells.

In some embodiments, a percentage of MDSCs as a proportion of live cellsis of one of fewer than 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6% or5% is predictive of increased and/or long-term survival, e.g. in asample obtained from a patient after completion of a therapeuticintervention (e.g. chemotherapy) to treat the disease, and prior toadministration of cells to the patient.

In some embodiments, a percentage of FoxP3+, CTLA4+ Tregs as aproportion of CD3+ cells is of one of fewer than 6%, 5.5%, 5%, 4.5%, 4%,3.5%, or 3% is predictive of increased and/or long-term survival, e.g.in a sample obtained from a patient prior to a therapeutic intervention(e.g. chemotherapy and/or ACT) to treat the disease.

Predicting Whether the Patient Will be a Long-Term Survivor on Treatmentby ACT

The methods of the present invention involve predicting, based on theanalysis of a blood-derived sample obtained from the patient, whetherthe patient will be a long-term survivor on treatment of the disease byACT.

In some embodiments, the patient is predicted to be a long-term survivoron treatment of the disease by ACT based on determination of one or morepositive predictors of increased and/or long-term survival as describedherein. In some embodiments, the patient is predicted to be a long-termsurvivor on treatment of the disease by ACT based on determination of 1,2, 3, 4, 5, 6, 7, 8, 9, 10 or more positive predictors of increasedand/or long-term survival. In some embodiments, the patient is predictedto be a long-term survivor on treatment of the disease by ACT based ondetermination of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 positivepredictors of increased and/or long-term survival.

Treatment of a Disease by ACT

The present invention provides methods of treating a patient by ACT, aform of immunotherapy. ACT may involve isolating at least one cell froma subject; treating the at least one cell, and; administering thetreated cell to a subject. In some embodiments, the subject from whichthe cell is isolated is the subject administered with the treated cell(i.e., adoptive transfer is of autologous cells). In some embodiments,the subject from which the cell is isolated is a different subject tothe subject to which the treated cell is administered (i.e., adoptivetransfer is of allogenic T cells).

In some embodiments, the treatment is to expand the number of cells. Insome embodiments, the treatment is to generate or expand the number of acell type which is effective to treat the disease. For example, wherethe disease is a disease caused or exacerbated by infection with EBV,the treatment of the cells may be to generate or expand the number ofEBV-specific cells (e.g. EBV-specific T cells, e.g. EBV-specific CTLs),which are then introduced into the patient. The treatment may beperformed in vitro. The treatment may comprise stimulation withEBV-infected cells, e.g. EBV-transformed lymphoblastoid cell line (LCL)cells.

Where the disease is a disease caused or exacerbated by infection withHPV, the treatment of the cells may be to generate or expand the numberof HPV-specific cells (e.g. HPV-specific T cells, e.g. HPV-specificCTLs), which are then introduced into the patient. The treatment may beperformed in vitro. The treatment may comprise stimulation withHPV-infected cells.

Methods for expanding virus-specific T cells are well known to theskilled person. Typical culture conditions (i.e. cell culture media,additives, temperature, gaseous atmosphere), ratios of responder cellsto stimulator cells, culture periods for stimulation steps, etc. can bereadily determined by reference e.g. to Bollard et al., J Exp Med(2004), 200(12): 1623-1633 and Straathof et al., Blood (2005), 105(5):1898-1904, both hereby incorporated by reference in entirety.

In some embodiments the method may comprise one or more of the followingsteps: taking a blood sample from a subject; isolating and/or expandingat least one T cell from the blood sample; culturing the at least one Tcell in in vitro or ex vivo cell culture; stimulating the at least one Tcell; collecting the at least one T cell; mixing the modified T cellwith an adjuvant, diluent, or carrier; administering the at least one Tcell to a subject.

The subject to be treated according to the invention may be any animalor human. The subject is preferably mammalian, more preferably human.The subject may be a non-human mammal, but is more preferably human. Thesubject may be male or female. The subject may be a patient. A subjectmay have been diagnosed with a disease or condition requiring treatment,may be suspected of having such a disease or condition, or may be atrisk from developing such a disease or condition.

Administration of cells according to the invention is preferably in a“therapeutically effective” or “prophylactically effective” amount, thisbeing sufficient to show benefit to the subject. The actual amountadministered, and rate and time-course of administration, will depend onthe nature and severity of the disease or disorder. Prescription oftreatment, e.g. decisions on dosage etc., is within the responsibilityof general practitioners and other medical doctors, and typically takesaccount of the disease/disorder to be treated, the condition of theindividual subject, the site of delivery, the method of administrationand other factors known to practitioners. Adoptive transfer of virusspecific T cells is described, for example, in Cobbold et al., (2005) J.Exp. Med. 202: 379-386 and Rooney et al., (1998), Blood 92:1549-1555,incorporated by reference hereinabove.

In some embodiments, the method comprises additional therapeutic orprophylactic intervention. In some embodiments, the therapeutic orprophylactic intervention is selected from chemotherapy, immunotherapy,radiotherapy, surgery, vaccination and/or hormone therapy. In someembodiments, the additional therapeutic or prophylactic intervention isperformed contemporaneously with treatment of the disease by ACT, and/ormay be performed before and/or after treatment of the disease by ACT.

In one aspect, the present invention provides a method of treating apatient by adoptive cell transfer (ACT), the method comprising:

-   -   (i) administering a dose of cells to a patient;    -   (ii) analysing a blood-derived sample obtained from the patient        for one or more prognostic markers of long-term survival on        treatment of a disease by ACT;    -   (iii) based on the analysis of step (ii), predicting long-term        survival of the patient by treatment of the disease by ACT; and    -   (iv) administering one or more further doses of cells to the        patient.

In some embodiments, the analysis/prediction of whether the patient willbe a long-term survivor on treatment of the disease by ACT influencesthe decision as to whether to treat, or continue to treat, the diseaseby ACT.

In some embodiments, the method is used to stratify patients for whichtreatment by ACT is predicted to be associated with long-term survival,as compared to patients for which treatment by ACT is not predicted tobe associated with long-term survival.

That is, the present invention may be employed to the identificationand/or selection of (i) a sub-population of patients suffering from adisease to be treated by ACT in which treatment by ACT is predicted tobe associated with long-term survival, and/or (ii) a sub-population ofpatients suffering from a disease to be treated by ACT in whichtreatment by ACT is predicted not to be associated with long-termsurvival.

Accordingly, in a related aspect the present invention provides a methodof selecting a patient for treatment of a disease by adoptive celltransfer (ACT), comprising:

-   -   (i) analysing a blood-derived sample obtained from the patient        for one or more prognostic markers of long-term survival on        treatment of a disease by ACT;    -   (ii) based on the analysis of step (i), predicting whether the        patient will be a long-term survivor on treatment of the disease        by ACT; and    -   (iii) selecting a patient predicted to be a long-term survivor        on treatment of the disease by ACT for treatment of the disease        by ACT.

In some embodiments, the method additionally comprises an initial stepof administering a dose of cells to a patient. The dose of cells may beadministered in accordance with a method of treatment of the disease byACT, or may be administered as an initial screening step to evaluate thelikely response of the patient to treatment of the disease by ACT.

In a further related aspect the present invention provides a method ofselecting a patient for continued treatment of a disease by adoptivecell transfer (ACT), comprising:

-   -   (i) administering a dose of cells to a patient in accordance        with treatment of a disease by ACT;    -   (ii) analysing a blood-derived sample obtained from the patient        for one or more prognostic markers of long-term survival on        treatment of a disease by ACT;    -   (iii) based on the analysis of step (ii), predicting whether the        patient will be a long-term survivor on treatment of the disease        by ACT; and    -   (iv) selecting a patient predicted to be a long-term survivor on        treatment of the disease by ACT for continued treatment of the        disease by ACT.

The method may further comprise step (v) administering one or more dosesof cells to patients selected in (iv).

Improving Long-Term Survival of a Patient on Treatment of the Disease byACT

The present invention also provides methods of treatment of a disease byACT, and/or methods for enhancing the effectiveness of treatment of adisease by ACT, comprising administering to a patient one or more agentsfor modifying the level of one or more prognostic markers of long-termsurvival on treatment of a disease by ACT.

In some embodiments, the methods comprise administering one or moreagents for decreasing MDSC number or activity and/or decreasingregulatory T lymphocyte number or activity, for increasing effector Tlymphocyte number or activity, and/or for reducing the amount of aninfectious agent associated with the disease.

In some embodiments, the one or more agents are administered to apatient after analysis in accordance with a method for predictingwhether a patient will be a long-term survivor on treatment of a diseaseby ACT, a method of treating a patient by ACT, or a method of selectinga patient for treatment of a disease by ACT as described herein. In someembodiments, the one or more agents are administered based on predictionthat the patient will not be a long-term survivor on treatment of thedisease by ACT.

In some embodiments, the one or more agents are administered prior toadministration of a dose of cells to the patient. In some embodimentsthe one or more agents are administered after a dose of cells has beenadministered to the patient. In some embodiments, the one or more agentsare administered contemporaneously with treatment of the disease by ACT.

Also provided by the present invention is an agent for use in a methoddescribed herein, use of an agent in the manufacture of a medicament foruse in a method described herein, and the use of an agent in a methoddescribed herein.

The skilled person is well able to identify agents for decreasing MDSCnumber or activity and/or decreasing regulatory T lymphocyte number oractivity, for increasing effector T lymphocyte number or activity,and/or for reducing the amount of an infectious agent associated withthe disease.

In some embodiments, the agent capable effect reduced or increasedgene/protein expression and/or function by influencing transcription,mRNA processing (e.g. splicing), mRNA stability, translation,post-translational processing, protein stability, protein degradationand/or protein function/activity. Examples of agents includeantagonist/agonist antigen-binding molecules, nucleic acids (e.g. siRNA,shRNA), small molecule inhibitors/agonists etc.

Kits

The present invention also provides kits for use in accordance with themethods described herein.

In some embodiments the kit may be suitable for analysing ablood-derived sample as described herein for one or more prognosticmarkers of long-term survival on treatment of a disease by ACT. The kitmay be employed to predict whether or not a patient will be a long-termsurvivor on treatment of the disease by ACT.

According the present invention provides a kit comprising means fordetecting a marker of MDSC number or activity and/or regulatory Tlymphocyte number or activity, and means for detecting a marker ofeffector T lymphocyte number or activity, optionally further comprisingmeans for detecting a marker of the amount of an infectious agentassociated with the disease.

The kit may have at least one container having a predetermined quantityof: one or more reagents for detecting the level of one or more markersof MDSC number or activity and/or one or more markers of regulatory Tlymphocyte number or activity, one or more reagents for detecting thelevel of one or more markers of effector T lymphocyte number oractivity, and/or one or more reagents for detecting the level of one ormore markers of the amount of an infectious agent associated with thedisease.

The skilled person is readily able to identify reagents suitable fordetermining the level of the relevant gene/protein expression oractivity in a sample. Suitable reagents may include e.g. oligonucleotideprimers, ELISA antibody pairs, aptamers etc. Suitable reagents mayinclude detectable labels for detection and quantification

In some embodiments, the kit contains all of the components necessaryand/or sufficient to perform an assay on a blood-derived sample forpredicting whether a patient will be a long-term survivor on treatmentof the disease by ACT, including all controls, instructions/directionsfor performing assays, and any necessary software for analysis andpresentation of results.

The invention includes the combination of the aspects and preferredfeatures described except where such a combination is clearlyimpermissible or expressly avoided.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

Aspects and embodiments of the present invention will now beillustrated, by way of example, with reference to the accompanyingfigures. Further aspects and embodiments will be apparent to thoseskilled in the art. All documents mentioned in this text areincorporated herein by reference.

Throughout this specification, including the claims which follow, unlessthe context requires otherwise, the word “comprise,” and variations suchas “comprises” and “comprising,” will be understood to imply theinclusion of a stated integer or step or group of integers or steps butnot the exclusion of any other integer or step or group of integers orsteps.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Ranges may be expressedherein as from “about” one particular value, and/or to “about” anotherparticular value. When such a range is expressed, another embodimentincludes from the one particular value and/or to the other particularvalue. Similarly, when values are expressed as approximations, by theuse of the antecedent “about,” it will be understood that the particularvalue forms another embodiment.

Examples

In the following Examples, the inventors describe analysis of samplescollected from a Phase H clinical trial in which Epstein-Barr virus(EBV)-positive nasopharyngeal carcinoma (NPC) patients were treated withgemcitabine and carboplatin for four cycles, followed by treatment byadoptive transfer of autologous, in vitro expanded, EBV-specific CD8+T-cells.

The inventors undertook a multifactorial analysis to determine systemicimmune sera correlates that influence and determine successful therapy,and examined the cellular immune environment using cryopreserved PBMCsfrom samples collected before and after chemotherapy, and throughoutimmunotherapy, to evaluate the immunosuppressive state of the patients.

The inventors demonstrate key differences between non-survivors andlong-term survivors in terms of Teffector:Treg ratios and whether or notthere is expansion or contraction of the myeloid-derived suppressor cell(MDSC) compartment.

Example 1: Experimental Procedures

1.1 Immunotherapy Trial and Sample Collection

A Phase II clinical trial was performed in which Epstein-Barr virus(EBV)-positive nasopharyngeal carcinoma (NPC) patients were treated withgemcitabine and carboplatin for four cycles, followed by treatment byadoptive transfer of autologous, in vitro expanded, EBV-specific CD8⁺T-cells.

The results demonstrated unprecedented efficacy. The two-year overallsurvival rate was 62.9%, and the three-year overall survival rate was37.1%. These two- and three-year overall survival rates are amongst thebest survival rates for treatment of advanced NPC.

Analysis of the Phase II trial data showed that there was a positivecorrelation between long-term survivors and the ability of their CTLs toproduce IFN-γ in response to LMP2 antigen presentation.

Throughout the immunotherapy trial peripheral blood was sampled from thepatients. The time points examined were:

-   -   Pre chemotherapy (T−1);    -   Post chemotherapy, pre-immunotherapy (T0);    -   Post first CTL infusion (T1);    -   Post second CTL infusion (T2); and    -   Post third CTL infusion (T3).

1.2 Serum Cytokine Analysis

A 27-plex human cytokine and chemokine luminex multiplex bead arrayassay kit (Invitrogen; Carlsbad, Calif.) was used to measure levels ofthe following cytokines in diluted plasma obtained from samples: IL-3,IL-4, IL-6, IL-8, IL-9, IL-10, IL-15, IL-17, IL-21, (CXCL10) IP-10, CCL3(MIP-1a), CCL4 (MIP-1β), CCL20 (MIP-3sa), MCP-1, IFN-α2, IFN-γ, EGF,FGF-2, VEGF, TGFA, CD40L, fractalkine, GM-CSF, G-CSF, GRO, MDC, andeotaxin.

Plates were washed using Biotek ELx405 washer (Biotek, USA) and ‘read’with Flexmap 3D systems (Luminex Corp, Austin, Tex., USA) as per themanufacturer's instructions. Data were analyzed using Bio-Plex manager6.0 software with a 5-parameter curve-fitting algorithm applied forstandard curve calculations.

1.3 Immunophenotyping of PBMCs

PBMCs obtained from frozen patient samples collected at the timepointsT−1 to T3 were stained with two different fluorescently labeledmonoclonal antibody panels (Treg panel or MDSC panel) to determine celllineage and activation status.

Treg panel: BUV 395 ant-CD25, Pacific Blue anti-FoxP3, BV 711 ant-CD127,FITC anti-CD4, PE anti-CTLA4, PECF594 anti-CD45RA, PECy5 anti-CD3, PECy7 anti-CCR7, near infrared live/dead cell stain.

Tregs were identified using a single cell gate, live/dead cell negative,CD3 positive, CD4 positive, CD25 positive, CD127 negative, FOXP3positive, CTLA4 positive gating strategy.

MDSC panel: BUV 395 anti-CD15, FITC anti-CD16, PE anti-CD33, PECF594anti-CD34, PE Cy7 anti-CD11b, APC anti-CD14, APC H7 anti-HLA-DR, violetlive/dead cell stain.

Monocytic MDSCs were identified using a single cell gate, live/dead cellnegative, CD16 negative, CD15 negative, CD34 negative, CD11 b positive,CD33 positive, CD14 intermediate, HLA-DR negative-low gating strategy.Cells were acquired using an LSRII (BD Biosciences) flow cytometer. Datawas analysed using Diva (BD Biosciences) and Flowjo (Treestar) software.

1.4 RNA Isolation

1×10⁵ thawed PBMCs from time points T−1, T0, T1, T2 and T3, werepelleted by centrifugation in eppendorf tubes. Samples subsequentlyunderwent RNA extraction using a QIAGEN RNAEasy Micro kit. Samples wereprocessed according to the manufacturer's guidelines. Final elutionvolume was in 15 μl RNase free water.

1.5 Nanostring Processing

Gene expression was analysed using a Nanostring PanCancer Immune Panel.100 ng of each patient sample was prepared as per the manufacture'sguidelines. Quantification of gene expression was determined using thenCounter platform, raw counts were processed using nSolver. Counts werenormalised using the ComBat method.

1.6 Bayesian Network Analysis

Bayesian networks of combined luminex and nanostring data for eachseparate time point, were calculated using BayesiaLab. Two-year survivalstatus was excluded from the initial network, which was formed using themaximum spanning tree algorithm (structural coefficient=1). Nodes werethen clustered using the variable clustering function. Taboo learning,with two-year survival included, was utilised to form the final network.

1.7 Statistical Analysis

Correlation with overall survival was evaluated by analysis usingSpearman's ranked correlation. Correlation with two-year survival wasanaylsed using two-way ANOVA with Bonferroni correction.

Example 2: Long-Term Survivors Experience Increased Lymphoid butDecreased Myeloid Numbers Post CTL Immunotherapy

Throughout the immunotherapy trial peripheral blood was sampled from thepatients. In order to assess the impact of immunotherapy analysis wasfocused on the time point two weeks after the first immunotherapyinfusion (T1; see FIG. 1).

The numbers of peripheral blood leukocytes at time point T1 wereassessed to detect any gross changes in the immune system as a result ofimmunotherapy. The results are shown in FIGS. 2A to 2E.

There was a significant negative correlation between the number ofleukocytes and overall survival (r=−0.43; FIG. 2A). Neutrophil numbers(as a proportion of leukocytes) were also significantly, negativelyassociated with survival, albeit with poor correlation (r=−0.32; FIG.2B). Conversely there was a significant positive correlation betweenlymphocyte numbers (as a proportion of leukocytes) and overall survival(r=0.46; FIG. 2C). No correlation was observed between monocyte number(as a proportion of leukocytes) and overall survival (FIG. 2D). Analysisof leukocyte numbers at time points before immunotherapy (T0) or beforechemotherapy (T−1) did not reveal a statistically significantcorrelation with overall survival (FIG. 3; Table 1).

Taken together, the results suggested that long-term survivorsexperience a shift in leukocyte composition within two weeks after thefirst immunotherapy injection, results in decreased numbers of myeloidcells and increased numbers of effector T lymphocytes. The results alsodemonstrate that this change in the immune cell population occurs as adirect result of immunotherapy, and is not attributable to chemotherapy.

Example 3: Successful CTL Immunotherapy Results in Increased IFNγ

In order to assess whether or not the increase in lymphocyte numbers wasalso influencing viral load and cytokine production, patient sera wasanalysed for cytokine production by luminex assay, and EBV DNA wasquantified by qPCR. The results of the analyses are shown in FIG. 4A to4F. The data shown in FIGS. 4A to 4D, 5, 6 and 7A and 7B is based onanalysis of serum obtained from samples isolated at time point T1, i.e.two weeks after the first infusion of EBV-specific CTL.

Within two weeks after the first immunotherapy infusion there was asignificant positive correlation between IFNγ production and survival(FIG. 4A). Prior to immunotherapy (i.e. at T0), there was no correlationwas observed between survival and IFNγ concentrations in the sera (Table2, FIG. 5). Conversely, survival was strongly correlated with asignificant decrease in EBV viral load (FIG. 4B). Production ofmyeloid-expressed chemokines such as CCL20 was similarly decreased inlong-term survivors (FIG. 4C). Whilst other cytokines produced bymyeloid cells such as CCL10 were not significantly associated withsurvival (FIG. 4D).

In order to investigate these correlations to serve as biomarkers forsurvival associated with treatment with autologous EBV-specific CTL,patients were sorted into groups of patients which survived for twoyears (Two Year Survivor) and patients which did not survive for twoyears (Non Survivor). Two-year survival was chosen as a cutoff due tothe fact that EBV+ Stage IV NPC patients have a median survival range of11-22 months.

Analysis of single analytes did not sufficiently stratify non-survivorsvs. two-year survivors (FIGS. 6A and 6B). The inventors thereforeanalysed ratios of the serum levels of several different cytokines andEBV DNA to the only cytokine which was positively correlated withsurvival, IFNγ. IFNγ was used as the consequent, and analytes whichcorrelated with non-survival were used as the antecedent.

The results are shown in FIGS. 7A and 7B. Using a combination of EBVDNA:IFNγ vs CCL20:IFNγ, or of EBV DNA:IFNγ vs CCL10:IFNγ, the inventorswere able to correctly identify two-year survivors with a true positiverate of 85%. Thus the inventors demonstrated the ability of the serumlevels of combinations of markers at two weeks after the commencement oftreatment with autologous EBV-specific CTL to predict long-term survivalof patients.

In order to validate the findings, the ratiometric values were inputtedinto the Weka machine learning software algorithm. The logisticregression methodology was also able to correctly classify patients witha success rate of 85%. To evaluate the effectiveness of the ratiosinstead of actual values, the same data was inputted replacing theratios with single analyte measurements. Removal of one of the analytesfrom the ratios reduced the correctly classified rate to 57%.

Together, the results show that successful immunotherapy induces greaterIFNγ production in long-term survivors, whilst the level of productionof chemokines produced by myeloid cells is reduced.

Example 4: Non-Survivors Express Increased Transcripts Associated withMyeloid Cells

The inventors next decided to investigate the reasons for therapeuticfailure in individuals who survived for less than two-years.

Patient PBMCs obtained at time point T1 were subjected to nanostringanalysis for transcript quantification. RNA counts were normalized,filtered for significance and subsequently analysed using a Bayesiannetwork (FIG. 8). Examination of this network revealed an enrichment oftranscripts associated with markers of myeloid cells and immuneinhibitory markers. Spearman's ranked correlation was used to furtherfilter and identify associations with therapeutic failure. The strongestcorrelations observed with overall survival were CD68, LILRA5, andS100A8 transcription, all of which are associated with myeloid cells andinhibitory marker expression (FIGS. 10A, 10B, and 10C).

In addition, CD8 transcription was also found to be significantlyassociated with overall survival (Table 3, FIG. 9).

FIG. 18A to 18C shows nanostring analysis of transcription of theinflammasome and inhibitory markers NLRP3, CD274 (PD-L1) and STAT4 attime points T−1 (pre-chemotherapy) and T0 (post-chemotherapy) fornon-survivors and long-term survivors.

FIGS. 19 to 21 show the results of further nanostring and Bayesiannetwork analysis of RNA obtained from patient PBMCs at time point T1.Examination of this network revealed an enrichment of transcriptsassociated with myeloid and inhibitory markers. Spearman's rankedcorrelation was used to further filter and identify associations withtherapeutic failure. The strongest correlations observed with overallsurvival were CD68, LILRA5, and S100A9 transcription, all of which areassociated with myeloid cells and inhibitory marker expression (FIGS.20A, 20B, and 20C). In addition, CD8B transcription was also found to besignificantly associated with overall survival (Table 4, FIG. 21).

Together, these results suggest that the presence of amyeloid/inhibitory leukocyte signature in the peripheral bloodnegatively impacts long-term patient survival, potentially by creatingan inhibitory environment which negatively influences CTL infusionefficacy.

Example 5: Non-Survivors Experience an Increase of Myeloid-DerivedSuppressor Cells Post Chemotherapy

In order to further examine the impact of this myeloid/inhibitorysignature on survival the inventors extended analysis of the whole bloodcounts to time points before immunotherapy (i.e. T−1 and T0), as it hasrecently been shown that successful T cell vaccination relies onablation of the myeloid compartment (Welters, M. J., van der Sluis, T.C., van Meir, H., Loof, N. M., van Ham, V. J., van Duikeren, S., et al.(2016). Vaccination during myeloid cell depletion by cancer chemotherapyfosters robust T cell responses. Science Translational Medicine, 8(334),334ra52-334ra52. http://doi.org/10.1126/scitranslmed.aad8307).

Bayesian network analysis of the post chemotherapy time-point (i.e. T0)revealed that CD68 expression was negatively correlated with two-yearsurvival. This suggests that increased presence of myeloid cellscontribute to decreased patient survival after immune cell ablation(FIG. 22; Table 5, FIG. 23).

Discretization of patients based on two-year survival (non-survivors andtwo-year survivors) showed that monocyte numbers (as a proportion ofleukocytes) were increased in both groups post-chemotherapy as comparedto before chemotherapy (i.e. at T0 as compared to T−1), with thenon-survivors showing the greater increase (FIG. 11A). These resultswere confirmed by flow cytometric analysis, however the rate of increasefor two-year survivors was far less than observed by clinical wholeblood counts (FIG. 11B). In both analyses, the number of monocytes inboth groups was similar at two weeks post first immunotherapy (T1).

Conversely, time course analysis of the neutrophil compartment bytwo-year survival showed severe decreases in the number of neutrophils(as a proportion of leukocytes), which were not statistically differentbetween the groups (FIG. 12A). Differences were also observed in thelymphocyte compartment, with two-year survivors having increased numbersof lymphocytes (as a proportion of leukocytes) throughout the timecourse as compared to non-survivors (FIG. 12B).

The inventors next examined the phenotype of the peripheral,cryopreserved leukocytes, focusing in particular on monocytic-MDSCs andregulatory T-cells (FIG. 13). The time points examined were prechemotherapy (T−1), post chemotherapy pre-immunotherapy (T0), post firstCTL infusion (T1), post second CTL infusion (T2), and post third CTLinfusion (T3).

The proportion of MDSC at different time points as a proportion of thetotal number of live cells is shown in FIGS. 14A and 14B. No differencein MDSC numbers was observed between two-year survivors andnon-survivors pre-chemotherapy (i.e. at T−1). After chemotherapeutictreatment at T0, a significant increase of MDSC numbers was observed inthe non-survivors, whilst the long-term survivors exhibited a small butnon-significant increase. Numbers of MDSCs in non-survivors thendecreased but remained at levels above the number of MDSCs in long-termsurvivors (T1). At later time points the two groups of patientsdisplayed comparable MDSC numbers, which were at levels below thepre-chemotherapy time point (FIGS. 14A and 14B). The results show thatindividuals who did not undergo successful immunotherapeutic treatmentpossess an increased number of monocytes, the majority of which werecomprised of monocytic-MDSCs, at the time of first CTL infusion. Theincreased MDSC numbers at the time of first infusion may provide aninhibitory environment, thereby inhibiting the function of the infusedCTLs.

Example 6: Long-Term Survivors with High MDSC Numbers Possess DecreasedNumbers of Activated Tregs

From the MDSC analysis it was noted that a subset of two-year survivorshave high numbers of MDSCs post chemotherapy (>5.12% of live cells; seee.g. FIG. 14B), yet these individuals still underwent successfulimmunotherapeutic treatment. In order to investigate the role of otherinhibitory leukocyte subsets that could account for this discrepancy,the cryopreserved PBMCs were also examined for regulatory T cell (Treg)numbers at different time points.

The results are shown in FIG. 15. The number of activated Tregs duringthe clinical trial was not significantly different between non-survivorsand two-year survivors. Non-survivors did however display greater numberof CTLA4+ Tregs before chemotherapy (i.e. at T−1). These numbers weredecreased in both groups post chemotherapy (T0), with the non-survivorspossessing slightly higher amounts of Tregs throughout the time courseexamined (FIG. 15). Examination of the subset of individuals with highnumbers of MDSCs revealed a negative correlation between long-termsurvival and Treg numbers at time point T0, which might account for thesuccessful therapy in the subset of two-year survivors having highnumbers of MDSCs (FIGS. 16A and 16B).

Taken together these results show patients who survived for two yearshad decreased numbers of MDSCs and Tregs post chemotherapy, whichallowed for the establishment potent CTL responses as evidenced byincreased levels of IFNγ.

Example 7: Data Across all Time Points

The levels of IFNγ, CCL22, IL-10, IL-8, CCL20 (i.e. MIP3α) and VEGFdetermined in samples obtained from non-survivors and long-termsurvivors (i.e. more than 2 years) detected in samples obtained frompatients at different time points over the course of the clinical trialwere amalgamated and analysed.

The results are shown in FIG. 17, and show that the level of IFNγpositively correlated with long-term survival, whilst levels of each ofCCL22, IL10, IL8, MIP3α and VEGF are negatively correlated withlong-term survival.

REFERENCES

-   1. Chang, C. M., Yu, K. J., Mbulaiteye, S. M., Hildesheim, A. &    Bhatia, K. The extent of genetic diversity of Epstein-Barr virus and    its geographic and disease patterns: a need for reappraisal. Virus    Res. 143, 209-221 (2009).-   2. Wee, J. et al. Randomized trial of radiotherapy versus concurrent    chemoradiotherapy followed by adjuvant chemotherapy in patients with    American Joint Committee on Cancer/International Union against    cancer stage III and IV nasopharyngeal cancer of the endemic    variety. J. Clin. Oncol. 23, 6730-6738 (2005).-   3. Gerdemann, U. et al. Nucleofection of DCs to generate    Multivirus-specific T cells for prevention or treatment of viral    infections in the immunocompromised host. Mol. Ther. 17, 1616-1625    (2009).-   4. Chia, W. K. et al. A phase II study evaluating the safety and    efficacy of an adenovirus-ΔLMP1-LMP2 transduced dendritic cell    vaccine in patients with advanced metastatic nasopharyngeal    carcinoma. Ann. Oncol. 23, 997-1005 (2012).-   5. Moosmann, A. et al. Effective and long-term control of EBV PTLD    after transfer of peptide-selected T cells. Blood 115, 2960-2970    (2010).-   6. Louis, C. U. et al. Enhancing the in vivo expansion of adoptively    transferred EBV-specific CTL with lymphodepleting CD45 monoclonal    antibodies in NPC patients. Blood 113, 2442-2450 (2009).-   7. Straathof, K. C. M. et al. Treatment of nasopharyngeal carcinoma    with Epstein-Barr virus-specific T lymphocytes. Blood 105, 1898-1904    (2005).-   8. Louis, C. U. et al. Adoptive transfer of EBV-specific T cells    results in sustained clinical responses in patients with    locoregional nasopharyngeal carcinoma. J. Immunother. 33, 983-990    (2010).-   9. Smith, C. et al. Effective treatment of metastatic forms of    Epstein-Barr virus-associated nasopharyngeal carcinoma with a novel    adenovirus-based adoptive immunotherapy. Cancer Res. 72, 1116-1125    (2012).-   10. Suzuki, E., Kapoor, V., Jassar, A. S., Kaiser, L. R. &    Albelda, S. M. Gemcitabine selectively eliminates splenic    Gr-1+/CD11b+ myeloid suppressor cells in tumor-bearing animals and    enhances antitumor immune activity. Clin. Cancer Res. 11, 6713-6721    (2005).-   11. Nowak, A. K., Robinson, B. W. S. & Lake, R. A. Gemcitabine    exerts a selective effect on the humoral immune response:    implications for combination chemo-immunotherapy. Cancer Res. 62,    2353-2358 (2002).-   12. Shevchenko, I. et al. Low-dose gemcitabine depletes regulatory T    cells and improves survival in the orthotopic Panc02 model of    pancreatic cancer. Int. J. Cancer 133, 98-107 (2013).-   13. Kan, S. et al. Suppressive effects of cyclophosphamide and    gemcitabine on regulatory T-cell induction in vitro. Anticancer Res    32, 5363-5369 (2012).-   14. Rettig, L. et al. Gemcitabine depletes regulatory T-cells in    human and mice and enhances triggering of vaccine-specific cytotoxic    T-cells. Int. J. Cancer 129, 832-838 (2011).-   15. Wesolowski, R., Markowitz, J. & Carson, W. E., Ill Myeloid    derived suppressor cells—a new therapeutic target in the treatment    of cancer. 1, 1-1 (2013).-   16. Dumitru, C. A., Moses, K., Trellakis, S., Lang, S. & Brandau, S.    Neutrophils and granulocytic myeloid-derived suppressor cells:    immunophenotyping, cell biology and clinical relevance in human    oncology. Cancer Immunol. Immunother. 61, 1155-1167 (2012).-   17. Filipazzi, P., Huber, V. & Rivoltini, L. Phenotype, function and    clinical implications of myeloid-derived suppressor cells in cancer    patients. Cancer Immunol. Immunother. 61, 255-263 (2011).-   18. Ding, Z.-C. et al. Immunosuppressive myeloid cells induced by    chemotherapy attenuate antitumor CD4+ T cell responses through the    PD-1/PD-L1 axis. Cancer Res.    (2014).doi:10.1158/0008-5472.CAN-13-3596-   19. Huang, A. et al. Increased CD14+HLA-DR−/low myeloid-derived    suppressor cells correlate with extrathoracic metastasis and poor    response to chemotherapy in non-small cell lung cancer patients.    Cancer Immunol. Immunother. 62, 1439-1451 (2013).

1-20. (canceled)
 21. A method of treating an Epstein-Barr Virus(EBV)-positive cancer in a patient by adoptive cell transfer (ACT) ofEBV-specific CTLs, the method comprising: (i) analysing a correlate ofmyeloid-derived suppressor cell (MDSC) number or activity in ablood-derived sample obtained from the patient; (ii) based on step (i),predicting whether the patient will be a long-term survivor on treatmentof the EBV-positive cancer by ACT of EBV-specific CTLs; and (iii)administering one or more doses of EBV-specific CTLs to a patientpredicted in step (ii) to be a long-term survivor on treatment of theEBV-positive cancer by ACT of EBV-specific CTLs.
 22. The methodaccording to claim 21, wherein the correlate of MDSC number or activityis selected from: the percentage of MDSCs amongst peripheral bloodmononuclear cells (PBMCs), the percentage of monocytes amongst PBMCs,and the percentage of monocytes amongst leukocytes.
 23. The methodaccording to claim 21, wherein analysing a correlate of MDSC number oractivity in a blood-derived sample obtained from the patient comprisesdetermining the percentage of MDSCs amongst peripheral blood mononuclearcells (PBMCs), the percentage of monocytes amongst PBMCs, or thepercentage of monocytes amongst leukocytes in a blood-derived sampleobtained from the patient, and comparing the determined percentage to areference value which is indicative of long-term survival ornon-survival of a patient on treatment of the EBV-positive cancer by ACTof EBV-specific CTLs.
 24. The method according to claim 21, wherein theblood derived-sample is obtained from the patient after completion of acourse of chemotherapy.
 25. The method according to claim 24, whereinthe chemotherapy comprises treatment with gemcitabine and/orcarboplatin.
 26. The method according to claim 21, wherein theEBV-positive cancer is selected from: EBV-positive nasopharyngealcarcinoma (NPC), EBV-positive liver cancer, EBV-positive lung cancer,and EBV-positive gastric cancer.
 27. A method of treating anEpstein-Barr Virus (EBV)-positive cancer in a patient by adoptive celltransfer (ACT) of EBV-specific CTLs, the method comprising: (i)analysing a correlate of myeloid-derived suppressor cell (MDSC) numberor activity in a blood-derived sample obtained from the patient; (ii)based on step (i), predicting whether the patient will be a long-termsurvivor on treatment of the EBV-positive cancer by ACT of EBV-specificCTLs; (iii) selecting a patient predicted to be a long-term survivor ontreatment of the EBV-positive cancer by ACT of EBV-specific CTLs fortreatment by ACT of EBV-specific CTLs; and (iv) administering one ormore doses of EBV-specific CTLs to the patient selected in step (iii).28. The method according to claim 27, wherein the correlate of MDSCnumber or activity is selected from: the percentage of MDSCs amongstperipheral blood mononuclear cells (PBMCs), the percentage of monocytesamongst PBMCs, and the percentage of monocytes amongst leukocytes. 29.The method according to claim 27, wherein analysing a correlate of MDSCnumber or activity in a blood-derived sample obtained from the patientcomprises determining the percentage of MDSCs amongst peripheral bloodmononuclear cells (PBMCs), the percentage of monocytes amongst PBMCs, orthe percentage of monocytes amongst leukocytes in a blood-derived sampleobtained from the patient, and comparing the determined percentage to areference value which is indicative of long-term survival ornon-survival of a patient on treatment of the EBV-positive cancer by ACTof EBV-specific CTLs.
 30. The method according to claim 27, wherein theblood derived-sample is obtained from the patient after completion of acourse of chemotherapy.
 31. The method according to claim 30, whereinthe chemotherapy comprises treatment with gemcitabine and/orcarboplatin.
 32. The method according to claim 27, wherein theEBV-positive cancer is selected from: EBV-positive nasopharyngealcarcinoma (NPC), EBV-positive liver cancer, EBV-positive lung cancer,and EBV-positive gastric cancer.
 33. A method of treating anEpstein-Barr Virus (EBV)-positive cancer in a patient by adoptive celltransfer (ACT) of EBV-specific CTLs, the method comprising: (i)administering a course of chemotherapy to the patient; (ii) analysing acorrelate of myeloid-derived suppressor cell (MDSC) number or activityin a blood-derived sample obtained from the patient; (iii) based on step(ii), predicting whether the patient will be a long-term survivor ontreatment of the EBV-positive cancer by ACT of EBV-specific CTLs; (iv)selecting a patient predicted to be a long-term survivor on treatment ofthe EBV-positive cancer by ACT of EBV-specific CTLs for treatment by ACTof EBV-specific CTLs; and (v) administering one or more doses ofEBV-specific CTLs to the patient selected in step (iv).
 34. The methodaccording to claim 33, wherein the correlate of MDSC number or activityis selected from: the percentage of MDSCs amongst peripheral bloodmononuclear cells (PBMCs), the percentage of monocytes amongst PBMCs,and the percentage of monocytes amongst leukocytes.
 35. The methodaccording to claim 33, wherein analysing a correlate of MDSC number oractivity in a blood-derived sample obtained from the patient comprisesdetermining the percentage of MDSCs amongst peripheral blood mononuclearcells (PBMCs), the percentage of monocytes amongst PBMCs, or thepercentage of monocytes amongst leukocytes in a blood-derived sampleobtained from the patient, and comparing the determined percentage to areference value which is indicative of long-term survival ornon-survival of a patient on treatment of the EBV-positive cancer by ACTof EBV-specific CTLs.
 36. The method according to claim 33, wherein thechemotherapy comprises treatment with gemcitabine and/or carboplatin.37. The method according to claim 33, wherein the EBV-positive cancer isselected from: EBV-positive nasopharyngeal carcinoma (NPC), EBV-positiveliver cancer, EBV-positive lung cancer, and EBV-positive gastric cancer.